U.S. patent application number 13/943011 was filed with the patent office on 2014-06-26 for beta 1,4-galactosyltransferases with altered donor and acceptor specificities, compositions and methods of use.
The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health & Human Servic, The United States of America, as represented by the Secretary, Department of Health & Human Servic. Invention is credited to Elizabeth Boeggeman, Pradman K. Qasba, Boopathy Ramakrishnan.
Application Number | 20140178985 13/943011 |
Document ID | / |
Family ID | 39203139 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178985 |
Kind Code |
A1 |
Qasba; Pradman K. ; et
al. |
June 26, 2014 |
BETA 1,4-GALACTOSYLTRANSFERASES WITH ALTERED DONOR AND ACCEPTOR
SPECIFICITIES, COMPOSITIONS AND METHODS OF USE
Abstract
The invention relates generally to beta
(1,4)-galactosyltransferase I mutants having altered donor and
acceptor specificities, and methods of use thereof. In addition,
the invention relates to methods for synthesizing oligosaccharides
using the beta (1,4)-galactosyltransferase I mutants and to using
the beta (1,4)-galactosyltransferase I mutants to conjugate agents,
such as therapeutic agents or diagnostic agents, to acceptor
molecules.
Inventors: |
Qasba; Pradman K.;
(Bethesda, MD) ; Ramakrishnan; Boopathy;
(Frederick, MD) ; Boeggeman; Elizabeth; (Bethesda,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health & Human Servic |
Rockville |
MD |
US |
|
|
Family ID: |
39203139 |
Appl. No.: |
13/943011 |
Filed: |
July 16, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12674655 |
Feb 22, 2010 |
8512991 |
|
|
PCT/US2007/018656 |
Aug 22, 2007 |
|
|
|
13943011 |
|
|
|
|
Current U.S.
Class: |
435/348 ;
435/252.31; 435/252.33; 435/252.34; 435/254.2; 435/320.1; 435/325;
435/352; 435/354; 435/356; 435/358; 435/365; 536/23.2 |
Current CPC
Class: |
A61P 7/02 20180101; A61P
37/04 20180101; C12P 21/005 20130101; C12N 9/1051 20130101; A61K
38/00 20130101 |
Class at
Publication: |
435/348 ;
536/23.2; 435/320.1; 435/252.33; 435/252.31; 435/252.34; 435/254.2;
435/358; 435/354; 435/352; 435/365; 435/325; 435/356 |
International
Class: |
C12N 9/10 20060101
C12N009/10 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Research supporting this application was carried out by the
United States of America as represented by the Secretary,
Department of Health and Human Services, This research was
supported by the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research. This research has
been funded in part with Federal funds from the National Cancer
Institute, NIH, under contract No. N01-C0-12400.
Claims
1-16. (canceled)
17. A nucleic acid molecule comprising SEQ ID NO: 1.
18. (canceled)
19. An expression cassette or vector comprising the nucleic acid of
claim 17.
20. (canceled)
21. A host cell comprising the expression cassette or vector of
claim 19 or 20.
22-137. (canceled)
138. The nucleic acid molecule of claim 17, wherein the nucleic
acid molecule encodes a polypeptide fragment of a beta
(1,4)-galactosyltransferase I that retains the ability to transfer
GalNAc or galactose from a sugar donor to a sugar acceptor.
139. The nucleic acid molecule of claim 17, wherein the nucleic
acid consists of SEQ ID NO: 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Divisional application of U.S. patent
application Ser. No. 12/674,655, filed Feb. 22, 2010, allowed,
which is a National Stage Entry of PCT/US07/18656, filed Aug. 22,
2007, each of which is hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to beta
(1,4)-galactosyltransferase I enzymes having altered donor and
acceptor specificities, and methods of use thereof. In addition,
the invention relates to methods for synthesizing oligosaccharides
using the beta (1,4)-galactosyltransferase I enzymes and to using
these beta (1,4)-galactosyltransferase I mutants to conjugate
agents, such as therapeutic agents or diagnostic agents, to
acceptor molecules.
BACKGROUND OF THE INVENTION
[0004] The present invention relates to the field of glycobiology,
specifically to glycosyltransferases, a superfamily of enzymes
involved in synthesizing carbohydrate moieties of glycoproteins,
glycolipids and glycosaminoglycans. The present invention provides
structure-based design of novel glycosyltransferases and their
biological applications.
[0005] Glycans can be classified as linear or branched sugars. The
linear sugars are the glycosaminoglycans comprising polymers of
sulfated disaccharide repeat units that are O-linked to a core
protein, forming a proteoglycan aggregate (Raman et al. 2005). The
branched glycans are found as N-linked and O-linked sugars on
glycoproteins or on glycolipids (Lowe et al., 2003). These
carbohydrate moieties of the linear and branched glycans are
synthesized by a super family of enzymes, the glycosyltransferases,
which transfer a sugar moiety from a sugar donor to an acceptor
molecule.
[0006] Eukaryotic cells express several classes of oligosaccharides
attached to proteins or lipids. Animal glycans can be N-linked via
beta-GlcNAc to Asn (N-glycans), O-linked via--GalNAc to Ser/Thr
(O-glycans), or can connect the carboxyl end of a protein to a
phosphatidylinositol unit (GPI-anchors) via a common core glycan
structure. Beta (1,4)-galactosyltransferase I catalyzes the
transfer of galactose from the donor, UDP-galactose, to an
acceptor, N-acetylglucosamine, to form a galactose-beta
(1,4)-N-acetylglucosamine bond, and thus allows galactose to be
linked to an N-acetylglucosamine that may itself be linked to a
variety of other molecules. Examples of such molecules include
other sugars and proteins. This reaction can be used to make many
types of molecules with biological significance. For example,
galactose-beta (1,4)-N-acetylglucosamine linkages are important for
many recognition events that control how cells interact with each
other in the body, and how cells interact with pathogens. In
addition, numerous other linkages of this type play a role in
cellular recognition and binding events, as well as in cellular
interactions with pathogens, such as viruses.
[0007] The structural information of glycosyltransferases has
revealed that the specificity of the sugar donor in these enzymes
is determined by a few residues in the sugar-nucleotide binding
pocket of the enzyme, which is conserved among the family members
from different species. This conservation has made it possible to
reengineer the existing glycosyltransferases with broader sugar
donor specificities. Mutation of these residues generates novel
glycosyltransferases that can transfer a sugar residue with a
chemically reactive functional group to N-acetylglucosarnine
(GlcNAc), galactose (Gal) and xylose residues of glycoproteins,
glycolipids and proteoglycans (glycoconjugates). Thus, there is
potential to develop mutant glycosyltransferases to produce
glycoconjugates carrying sugar moieties with reactive groups that
can be used in the assembly of bio-nanoparticles to develop
targeted-drug delivery systems or contrast agents for medical
uses.
[0008] Accordingly, methods to synthesize N-acetylglucosamine
linkages have many applications in research and medicine, including
in the development of pharmaceutical agents and improved vaccines
that can be used to treat disease.
SUMMARY OF THE INVENTION
[0009] As described below, the present invention describes a double
mutant beta 1,4galactosyltransferase, human
beta-1,4-Tyr285Leu-Met340His-Gal-T1, constructed from the
individual mutants, Tyr285Leu-Gal-T1 and Met340His-Gal-T1, that
transfers modified galactose in the presence of magnesium ion. The
invention is based on the structure-based design of beta
1,4-Galactosyltransferase I (beta4Gal-TI) that is dependent on
magnesium for its activity, and is equally efficient as
N-Acetylgalactosaminyltransferase in activity. The invention is
based further on the novel finding that the point mutations Tyr 285
to Len 285 and Met 340 to His340 of human beta Gal-T1
(Gal-T1-Y285L-M34014), changes the metal requirement from manganese
to magnesium and broadens the sugar donor specificity towards
N-acetylgalactosamine and 2'-modified galactose. In particular, the
invention describes a double mutant that can transfer galactose and
GalNAc as efficiently from its UDP-derivatives in the presence of
magnesium.
[0010] Accordingly, in a first aspect the invention provides a
polypeptide fragment of a beta (1,4)-galactosyltransferase I that
retains the ability to transfer GalNAc or galactose from a sugar
donor to a sugar acceptor in the presence of magnesium.
[0011] In one embodiment, the polypeptide fragment comprises an
amino acid substitution at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I (SEQ ID
NO: 2). In another embodiment, the polypeptide fragment comprises a
conservative amino acid substitution at amino acid positions 289
and 344 corresponding to bovine beta (1,4)-galactosyltransferase I
(SEQ ID NO: 2).
[0012] In a related embodiment, a leucine (L) is substituted for a
tyrosine (Y) at amino acid position 285 and a methionine (M) is
substituted for a histidine (H) at amino acid position 340 (SEQ ID
NO: 3). In another related embodiment, a leucine (L) is substituted
for a tyrosine (Y) at amino acid position 289 and a methionine (M)
is exchanged for a histidine (H) at amino acid position 344 (SEQ ID
NO: 4).
[0013] In another aspect, the invention provides a polypeptide
fragment of a beta (1,4)-galactosyltransferase I that retains the
ability to transfer GalNAc or galactose from a sugar donor to a
sugar acceptor, wherein the polypeptide fragment comprises SEQ ID
NO: 2.
[0014] In one embodiment of the above aspects, the sugar donor is
selected from UDP-GalNAc, UDP-galactose, UDP-GalNAc analogues or
UDP-galactose analogues. In another embodiment, the UDP-galactose
analogue or the UDP-GalNAc analogue comprises an azido group, a
keto group, or a thiol group. In a related embodiment, the azido
group, the keto group or the thiol group is substituted at the C2
position of galactose. In another related embodiment, one or more
agents are linked to a sugar moiety of the sugar donor.
[0015] In another embodiment, the agent is selected from the group
consisting of: antibodies, single chain antibodies, bacterial
toxins, growth factors, therapeutic agents, targeting agents,
contrast agents, chemical labels, a radiolabels, and fluorescent
labels.
[0016] In a related embodiment, the transfer occurs in the presence
of magnesium.
[0017] In still another related embodiment, the sugar acceptor is
N-acetylglucosamine (GlcNAc). In a further embodiment, the
N-acetylglucosamine (GlcNAc) is free or attached to a peptide of a
glycopeptide.
[0018] In another aspect, the invention provides a polypeptide
fragment from a beta 1,4-galactosyltransferase I that catalyzes the
formation of a GalNAc-beta-1,4-N-acetylgalactosamine bond in the
presence of magnesium.
[0019] In one embodiment, the polypeptide fragment comprises SEQ ID
NO: 2.
[0020] In another aspect, the invention provides a nucleic acid
molecule comprising SEQ ID NO: 1.
[0021] In still another aspect, the invention provides an isolated
amino acid sequence corresponding to the polypeptide fragment of
claim 15 comprising SEQ ID NO: 2.
[0022] In one embodiment, an expression cassette or vector
comprises a nucleic acid molecule described herein.
[0023] In another aspect, the invention features an expression
cassette or vector comprising a nucleic acid segment encoding a
polypeptide fragment of a beta (1,4)-galactosyltransferase I that
transfers GalNAc or galactose from a sugar donor to a sugar
acceptor, wherein the sugar donor comprises UDP-GalNAc,
UDP-Galactose, UDP-GalNAc analogue or a UDP-Galactose analogue, in
the presence of magnesium or that catalyzes the formation of a
GalNAc- or Gal-beta-1,4-N-acetylgalactosamine bond in the presence
of magnesium.
[0024] In one embodiment, a host cell comprises a expression
cassette or vector as described herein.
[0025] In another aspect, the invention features a method of making
a glycoprotein comprising incubating a reaction mixture comprising
a polypeptide fragment from a beta (1,4)-galactosyltransferase I
with a sugar donor and a sugar acceptor in the presence of
magnesium.
[0026] In one embodiment, the polypeptide fragment comprises an
amino acid exchange at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I (SEQ ID
NO: 3).
[0027] In another embodiment, the polypeptide fragment comprises a
conservative amino acid exchange at amino acid positions 289 and
344 corresponding to bovine beta (1,4)-galactosyltransferase (SEQ
ID NO: 4).
[0028] In another embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 285 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 340 of (SEQ ID
NO: 3).
[0029] In a further embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 289 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 344 of (SEQ ID
NO: 4).
[0030] In another aspect, the invention features a method of making
a glycoprotein comprising incubating a reaction mixture comprising
a polypeptide fragment from a beta (1,4)-galactosyltransferase I,
wherein the polypeptide fragment comprises SEQ ID NO: 2, with a
sugar donor and an sugar acceptor.
[0031] In one embodiment, the sugar donor is selected from
UDP-GalNAc, UDP-galactose, UDP-GalNAc analogues or UDP-galactose
analogues.
[0032] In another embodiment, the UDP-galactose analogue or
UDP-GalNAc analogue comprises an azido group, a keto group, or a
thiol group.
[0033] In another embodiment, the azido group, the keto group or
the thiol group is substituted at the C2 position of galactose.
[0034] In yet another embodiment, the one or more agents are linked
to a sugar moiety of the sugar donor.
[0035] In one embodiment, the agent is selected from the group
consisting of: antibodies, single chain antibodies, bacterial
toxins, growth factors, therapeutic agents, contrast agents,
targeting agents, chemical labels, a radiolabels, and fluorescent
labels.
[0036] In a particular embodiment, the transfer occurs in the
presence of magnesium.
[0037] In another particular embodiment, the sugar acceptor is
N-acetylglucosamine (GlcNAc), in a further embodiment, the
N-acetylglucosamine (GlcNAc) is free or attached to a peptide of a
glycopeptide.
[0038] In another aspect, the invention features a method of making
a glycoprotein comprising incubating a reaction mixture comprising
a polypeptide fragment of a beta 1,4-galactosyltransferase I that
catalyzes the formation of a GalNAc beta-1,4-N-acetylgalactosamine
bond with a sugar donor, in the presence of magnesium.
[0039] In one embodiment, the polypeptide fragment comprises SEQ ID
NO: 2.
[0040] In another aspect, the invention features a method to of
making a glycoprotein comprising incubating a reaction mixture
comprising a polypeptide fragment from a beta
(1,4)-galactosyltransferase I, wherein the polypeptide fragment
comprises SEQ ID NO: 2, with a sugar donor, wherein the sugar donor
comprises a UDP-galactose, UDP-GalNAc, UDP-GalNAc analogue or a
UDP-Gal analogue, and a N-acetylglucosamine sugar acceptor in the
presence of magnesium.
[0041] In still another aspect, the invention features an isolated
glycoprotein synthesized by the method comprising incubating a
reaction mixture comprising a polypeptide fragment from a beta
(1,4)-galactosyltransferase I with a sugar donor and a sugar
acceptor in the presence of magnesium.
[0042] In one embodiment, the polypeptide fragment comprises an
amino acid exchange at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I (SEQ ID
NO: 3).
[0043] In one embodiment, the polypeptide fragment comprises a
conservative amino acid exchange at amino acid positions 289 and
344 corresponding to bovine beta (1,4)-galactosyltransferase (SEQ
ID NO: 4).
[0044] In another embodiment, leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 285 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 340 of (SEQ
NO: 3).
[0045] In a further embodiment, a leucine (L) is substituted for a
tyrosine (Y) at amino acid position 289 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 344 of (SEQ ID
NO: 4).
[0046] In another aspect, the invention features a glycoprotein
synthesized by a method comprising incubating a reaction mixture
comprising a polypeptide fragment of a beta
(1,4)-galactosyltransferase I, wherein the polypeptide fragment
comprises SEQ ID NO: 2, with a sugar donor and an sugar
acceptor.
[0047] In one embodiment, the sugar donor is selected from
UDP-GalNAc, galactose, UDP-GalNAc analogues or UDP-galactose
analogues. In a further embodiment, the UDP-GalNAc or UDP-galactose
analogue comprises an azido group, a keto group, or a thiol group.
In a related embodiment, the azido group, the keto group or the
thiol group is substituted at the C2 position of galactose.
[0048] In another embodiment, one or more agents are linked to a
sugar moiety of the sugar donor. In a related embodiment, the agent
is selected from the group consisting of: antibodies, single chain
antibodies, bacterial toxins, growth factors, therapeutic agents,
contrast agents, targeting agents, chemical labels, a radiolabels,
and fluorescent labels.
[0049] In one embodiment, the transfer occurs in the presence of
magnesium.
[0050] In another embodiment, the sugar acceptor is
N-acetylglucosamine (GlcNAc). In a further embodiment, the
N-acetylglucosamine (GlcNAc) is free or attached to a peptide of a
glycopeptide.
[0051] In another aspect, the invention features a glycoprotein
synthesized by the method comprising incubating a reaction mixture
comprising a polypeptide fragment from a beta
1,4-galactosyltransferase I that catalyzes the formation of a
GalNAc- or beta-1,4-N-acetylgalactosamine bond in the presence of
magnesium.
[0052] In one embodiment, the polypeptide fragment comprises SEQ ID
NO: 2.
[0053] In another aspect, the invention features a glycoprotein
synthesized by the method comprising incubating a reaction mixture
comprising a polypeptide fragment from a beta
(1,4)-galactosyltransferase I, wherein the polypeptide fragment
comprises SEQ ID NO: 1, with a sugar donor, wherein the sugar donor
comprises UDP-GalNAc, or a UDP-GalNAc analogue, and a
N-acetylglucosamine sugar acceptor in the presence of
magnesium.
[0054] In another aspect, the invention features a composition
comprising a polypeptide fragment of a beta
(1,4)-galactosyltransferase I that transfers GalNAc or galactose
from a sugar donor to a sugar acceptor in the presence of
magnesium.
[0055] In one embodiment, the polypeptide fragment comprises an
amino acid substitution at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I (SEQ ID
NC): 3).
[0056] In another embodiment, the polypeptide fragment comprises a
conservative amino acid substitution at amino acid positions 289
and 344 corresponding to bovine beta (1,4)-galactosyltransferase I
(SEQ ID NO: 4).
[0057] In another embodiment, a leucine (L) is substituted for a
tyrosine (Y) at amino acid position 285 and a methionine (M) is
substituted for a histidine (H) at amino acid position 340 of (SEQ
ID NO: 3).
[0058] In another embodiment, a leucine (L) is substituted for a
tyrosine (Y) at amino acid position 289 and a methionine (M) is
substituted for a histidine (H) at amino acid position 344 of (SEQ
ID NO: 4).
[0059] In another aspect, the invention features a composition
comprising a polypeptide fragment from a beta
(1,4)-galactosyltransferase I that transfers GalNAc or galactose
from a sugar donor to a sugar acceptor, wherein the polypeptide
fragment comprises SEQ ID NO: 2.
[0060] In one embodiment, the sugar donor is selected from
UDP-GalNAc, UDP-galactose, UDP-GalNAc analogue or UDP-galactose
analogue.
[0061] In another embodiment, the UDP-GalNAc analogue or the
UDP-galactose analogue comprises an azido group, a keto group, or a
thiol group. In another embodiment, the azido group, the keto group
or the thiol group is substituted at the C2 position of
galactose.
In another embodiment, one or more agents are linked to a sugar
moiety of the sugar donor. In another related embodiment, the agent
is selected from the group consisting of: antibodies, single chain
antibodies, bacterial toxins, growth factors, therapeutic agents,
targeting agents, contrast agents, chemical labels, a radiolabels,
and fluorescent labels.
[0062] In one embodiment, the transfer occurs in the presence of
magnesium.
[0063] In another embodiment, the sugar acceptor is
N-acetylglucosamine (GlcNAc).
[0064] In another embodiment, the N-acetylglucosamine (GlcNAc) is
free or attached to a peptide of a glycopeptide.
[0065] In another aspect, the invention features a composition
comprising a polypeptide fragment of a beta
1,4-galactosyltransferase I that catalyzes the formation of a
GalNAc-beta-1,4-N-acetylgalactosamine bond in the presence of
magnesium.
[0066] In one embodiment, the polypeptide fragment comprises SEQ ID
NO: 2.
[0067] In another embodiment, the composition further comprises an
adjuvant.
[0068] In another aspect, the invention features a method of
coupling an agent to a carrier protein comprising incubating a
reaction mixture comprising a polypeptide fragment of a beta
(1,4)-galactosyltransferase I, wherein the polypeptide fragment
comprises SEQ ID NO: 2, with a sugar donor, and a carrier protein,
in the presence of magnesium.
[0069] In one embodiment, the sugar donor is a UDP-GalNAc analogue
or a UDP-galactose analogue.
[0070] In another embodiment, the UDP-GalNAc analogue or the
UDP-galactose analogue comprises an azido group, a keto group, or a
thiol group. In a related embodiment, the azido group, the keto
group or the thiol group is substituted at the C2 position of
galactose.
[0071] In another embodiment, one or more agents are linked to a
sugar moiety of the sugar donor. In a related embodiment, the agent
is selected from the group consisting of: antibodies, single chain
antibodies, bacterial toxins, growth factors, therapeutic agents,
targeting agents, contrast agents, chemical labels, a radiolabels,
and fluorescent labels.
[0072] In one embodiment, the carrier protein is ovalbumin. In
another embodiment, the carrier protein is an IgG.
[0073] In another embodiment, the method comprises the steps of
coupling the C2 UDP-galactose analogue to biotin for detection.
[0074] In another further embodiment, the detection is by
chemiluminescent assay, in a related embodiment, the contrast agent
is a paramagnetic contrast agent. In another related embodiment,
the paramagnetic contrast agent is used in magnetic resonance
imaging.
[0075] In another aspect, the invention features a method for the
diagnosis or treatment of a subject having a disease or disorder
comprising administering to the subject an effective amount of
polypeptide fragment synthesized by the method comprising
incubating a reaction mixture comprising an polypeptide fragment
from a beta (1,4)-galactosyltransferase I with a sugar donor,
wherein one or more agents are linked to the sugar donor, and an
sugar accept or thereby diagnosing or treating the subject.
[0076] In one embodiment, the polypeptide fragment comprises an
amino acid exchange at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I (SEQ ID
NO: 3).
[0077] In another embodiment, the polypeptide fragment comprises a
conservative amino acid exchange at amino acid positions 289 and
344 corresponding to bovine beta (1,4)-galactosyltransferase I (SEQ
ID NO: 4).
[0078] In another embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 285 and a methionine (M) is
exchanged for a histidine (14) at amino acid position 340 of (SEQ
ID NO: 3).
[0079] In another embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 289 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 344 of (SEQ ID
NO: 4).
[0080] In another aspect, the invention features a method for the
diagnosis or treatment of a subject suffering from a disease or
disorder comprising administering to the subject an effective
amount of a polypeptide fragment synthesized by a method comprising
incubating a reaction mixture comprising a polypeptide fragment of
a beta (1,4)-galactosyltransferase I, wherein the polypeptide
fragment comprises SEQ ID NO: 2, with a sugar donor, wherein the
sugar donor comprises UDP-Galactose analogue and wherein one or
more agents are linked to the sugar donor, and an sugar acceptor in
the presence of magnesium, thereby diagnosing or treating the
subject.
[0081] In one embodiment, the sugar donor is a UDP-GalNAc analogue
or a UDP-galactose analogue. In another embodiment, the UDP-GalNAc
analogue or the UDP-galactose analogue comprises an azido group, a
keto group, or a thiol group. In a related embodiment, the azido
group, the keto group or the thiol group is substituted at the C2
position of galactose.
[0082] In another embodiment, one or more agents are linked to a
sugar moiety of the sugar donor. In a related embodiment, the agent
is selected from the group consisting of: antibodies, single chain
antibodies, bacterial toxins, growth factors, therapeutic agents,
targeting agents, contrast agents, chemical labels, a radiolabels,
and fluorescent labels.
[0083] In a further embodiment, the transfer occurs in the presence
of magnesium.
[0084] In another embodiment, the sugar acceptor is
N-acetylglucosamine (GlcNAc). In a related embodiment, the
N-acetylglucosamine (GlcNAc) is free or attached to a peptide of a
glycopeptide.
[0085] In another aspect, the invention features a method for the
diagnosis or treatment of a subject suffering from a disease or
disorder comprising administering to the subject an effective
amount of a polypeptide fragment synthesized by a method comprising
incubating a reaction mixture comprising a polypeptide fragment of
a beta 1,4-galactosyltransferase I that catalyzes the formation of
a GlcNAc-acetylgalactosamine bond in the presence of magnesium and
a sugar donor, wherein the sugar donor comprises a UDP-GalNAc or a
UDP-GalNAc analogue, and wherein one or more agents are linked to
the sugar donor.
[0086] In one embodiment, the polypeptide fragment comprises SEQ ID
NO: 2.
[0087] In another embodiment, at least two agents are linked to the
sugar donor. In a further embodiment, the agents comprise at least
a targeting agent and a therapeutic agent.
[0088] In another aspect, the invention features a method for
imaging a target cell or tissue in a subject comprising
administering to a subject a polypeptide fragment synthesized by a
method comprising incubating a reaction mixture comprising a
polypeptide fragment of a beta (1,4)-galactosyltransferase I with a
sugar donor, wherein one or more imaging agents are linked to the
sugar donor, and an sugar acceptor and thereby imaging a target
cell or tissue.
[0089] In one embodiment, the polypeptide fragment comprises an
amino acid exchange at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I (SEQ ID
NO: 3).
[0090] In another embodiment, the polypeptide fragment comprises a
conservative amino acid exchange at amino acid positions 289 and
344 corresponding to bovine beta (1,4)-galactosyltransferase I (SEQ
ID NO: 4).
[0091] In another embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 285 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 340 of (SEQ ID
NO: 3).
[0092] In another embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 289 and a methionine (M) is
exchanged Dora histidine (H) at amino acid position 344 of (SEQ ID
NO: 4).
[0093] In another aspect, the invention features a method for
imaging a target cell or tissue comprising administering to a
subject a polypeptide fragment synthesized by a method comprising
incubating a reaction mixture comprising a polypeptide fragment of
a beta (1,4)-galactosyltransferase wherein the polypeptide fragment
comprises SEQ ID NO: 2, with a sugar donor, and wherein one or more
imaging agents are linked to the sugar donor, and an sugar acceptor
in the presence of magnesium, thereby imaging a target cell or
tissue.
[0094] In one embodiment, the sugar donor is a UDP-GalNAc analogue
or a UDP-galactose analogue.
[0095] In another embodiment, the UDP-GalNAc analogue IMP-galactose
analogue comprises an azido group, a keto group, or a thiol
group.
[0096] In another embodiment, the azido group, the keto group or
the thiol group is substituted at the C2 position of galactose. In
a related embodiment, one or more imaging agents are linked to a
sugar moiety of the sugar donor. In another embodiment, the imaging
agent is selected from the group consisting of: chemical labels,
radiolabels, and fluorescent labels.
[0097] In another embodiment, the transfer occurs in the presence
of magnesium.
[0098] In another embodiment, the sugar acceptor is
N-acetylglucosamine (GlcNAc), in a related embodiment, the
N-acetylglucosamine (GlcNAc) is free or attached to a peptide of a
glycopeptide.
[0099] In another aspect, the invention features a method for
imaging a target cell or tissue comprising administering to a
subject a polypeptide fragment synthesized by a method comprising
incubating a reaction mixture comprising a polypeptide fragment of
a beta 1,4-galactosyltransferase I that catalyzes the formation of
a GlcNAc-beta-1,4-N-acetylgalactosamine bond in the presence of
magnesium and a sugar donor, wherein the sugar donor comprises a
UDP-galactose analogue, and wherein one or more imaging agents are
linked to the sugar donor.
[0100] In one embodiment, the polypeptide fragment comprises SEQ ID
NO: 2.
[0101] In another aspect, the invention features a method for
preventing platelet aggregation comprising administering to a
subject an effective amount of a polypeptide fragment synthesized
by the method comprising incubating a reaction mixture comprising a
polypeptide fragment from a beta (1,4)-galactosyltransferase with a
sugar donor and an sugar acceptor and thereby preventing platelet
aggregation.
[0102] In one embodiment, the polypeptide fragment comprises an
amino acid exchange at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I (SEQ ID
NO: 3).
[0103] In another embodiment, the polypeptide fragment comprises a
conservative amino acid exchange at amino acid positions 289 and
344 corresponding to bovine beta (1,4)-galactosyltransferase I (SEQ
ID NO: 4).
[0104] In another embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 285 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 340 of (SEQ ID
NO: 3).
[0105] In another embodiment, a leucine (L) is exchanged for a
tyrosine (Y) at amino acid position 289 and a methionine (M) is
exchanged for a histidine (H) at amino acid position 344 of (SEQ ID
NO: 4).
[0106] In another aspect, the invention features a method for
preventing platelet aggregation comprising administering to a
subject an effective amount of a polypeptide fragment synthesized
by the method comprising incubating a reaction mixture comprising a
polypeptide fragment of a beta (1,4)-galactosyltransferase I,
wherein the polypeptide fragment comprises SEQ ID NO: 2, with a
sugar donor, wherein the sugar donor comprises a UDP-GalNAc
analogue or a UDP-galactose analogue and a sugar acceptor in the
presence of magnesium, and thereby preventing platelet
aggregation.
[0107] In one embodiment, the sugar donor is a UDP-GalNAc analogue
or a UDP-galactose analogue.
[0108] In another embodiment, the sugar acceptor is
N-acetylglucosamine (GlcNAc).
[0109] In another embodiment, the N-acetylglucosamine (GlcNAc) is
free or attached to a peptide of a glycopeptide.
[0110] In another aspect, the invention features a method for
preventing platelet aggregation comprising administering to the
subject an effective amount of an isolated glycoprotein synthesized
by the method comprising incubating a reaction mixture comprising
an polypeptide fragment from a beta 1,4-galactosyltransferase I
that catalyzes the formation of a
GlcNAc-beta-1,4-N-acetylgalactosamine bond and a sugar donor,
wherein the sugar donor comprises UDP-GalNAc or a UDP-GalNAc
analogue.
[0111] In one embodiment, the polypeptide fragment comprises SEQ ID
NO: 2.
[0112] In another embodiment, the subject is suffering from
abnormal platelet aggregation caused by a genetic lesion.
[0113] In another embodiment, the subject is suffering from
abnormal platelet aggregation caused by a drug treatment.
[0114] In another aspect, the invention features a method for
inducing an immune response in a subject comprising administering
to the subject a composition as described in the aspects
herein.
[0115] In another aspect, the invention features a kit comprising
packaging material, and a polypeptide fragment from a beta
(1,4)-galactosyltransferase I according to any one of the
above-mentioned aspects.
[0116] In one embodiment, the kit further comprises a sugar
donor.
[0117] In another embodiment, the donor is selected from the group
consisting of UDP-galactose, UDP-GalNAc, UDP-GalNAc analogues or
UDP-Galactose analogues.
[0118] In another embodiment, an agent is linked to the sugar
donor.
[0119] In a further embodiment, the agent is selected from the
group consisting of: antibodies, single chain antibodies, bacterial
toxins, growth factors, therapeutic agents, contrast agents,
targeting agents, chemical labels, a radiolabels, and fluorescent
labels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0120] FIG. 1 (A-C) shows the MALDI mass spectra of glycans after
the transfer of GalNAc (B) or 2-keto-galactose (C) to the sugar
acceptor, heptasaccharide tetrapeptide (A),
Arg-[GlcNAc.beta.1,2-Man.alpha.1,6-(GlcNAc.beta.1,2-Man.alpha.1,3)-Man.be-
ta.1,4-GlcNAc.beta.1,4-GlcNAc.beta.]-Asn-Glu-Gly, by the double
substitution enzyme, human beta 4Gal-T1M340H_Y285L. Major peaks are
annotated with the carbohydrate structure shown in the symbols for
monosaccharides, according to the nomenclature adopted by the
consortium for functional glycomics, (on the world wide web at
functionalglycomics.org/static/consortium/). GlcNAc (squares),
mannose (spheres).
[0121] FIG. 2 is a graph showing specific activities of the
catalytic domain of the human beta 4Gal-T1-M340H-Y285L. Reactions
were performed under saturating conditions of all substrates. In
these reactions the concentrations for the donors UDP-galactose or
UDP-GalNAc was 500 .mu.M and the acceptor .beta.-benzyl-GlcNAc was
at 25 mM.
[0122] FIGS. 3A-3B show the catalytic domain corresponding to amino
acids 127-398 of the double substitution human
beta-4Gal-T1-M340H-Y285L. FIG. 3A shows the DNA sequence
corresponding to the protein sequence, amino acid residues 127 to
398 (SEQ ID NO: 1), FIG. 3B shows the protein sequence
corresponding to amino acid residues 127 to 398 (SEQ ID NO: 2),
[0123] FIG. 4 shows the amino acid sequence corresponding to human
beta 1,4 galactosyltransferase-I (SEQ ID NO: 3).
[0124] FIG. 5 shows the amino acid sequence corresponding to bovine
beta 1,4 galactosyltransferase-I (SEQ ID NO: 4).
[0125] FIG. 6 shows the nucleotide sequence corresponding to human
beta 1,4 galactosyltransferase-I (SEQ ID NO: 5).
[0126] FIG. 7 shows the amino acid sequence corresponding to human
beta 1,4 galactosyltransferase-I (SEQ ID NO: 6).
DETAILED DESCRIPTION OF THE INVENTION
[0127] The invention generally features beta
(1,4)-galactosyltransferase I enzymes having altered donor and
acceptor specificities, and methods of use thereof. In addition,
the invention relates to methods for synthesizing oligosaccharides
using the beta (1,4)-galactosyltransferase I enzymes as described
herein and to using the beta (1,4)-galactosyltransferase I enzymes
to conjugate agents, such as therapeutic agents or diagnostic
agents, to acceptor molecules. The glycoconjugates have use in, for
example, disease treatment, diagnostics, and imaging.
DEFINITIONS
[0128] The invention is better understood with the aid of the
following definitions.
[0129] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). As used herein, the following terms have the
meanings ascribed to them below, unless specified otherwise.
[0130] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof. The term "a
nucleic acid molecule" includes a plurality of nucleic acid
molecules.
[0131] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude other elements. "Consisting essentially of", when
used to define compositions and methods, shall mean excluding other
elements of any essential significance to the combination. Thus, a
composition consisting essentially of the elements as defined
herein would not exclude trace contaminants from the isolation and
purification method and pharmaceutically acceptable carriers, such
as phosphate buffered saline, preservatives, and the like.
"Consisting of" shall mean excluding more than trace elements of
other ingredients and substantial method steps for administering
the compositions of this invention. Embodiments defined by each of
these transition terms are within the scope of this invention.
[0132] The term "acceptor" is meant to refer to a molecule or
structure onto which a donor is actively linked through action of a
of a galactosyltransferase, or fragment thereof. Examples of
acceptors include, but are not limited to, carbohydrates,
glycoproteins, glycolipids. In preferred embodiments, the acceptor
polypeptide can comprise, N-acetylglucosamine (GlcNAc) residues,
free or attached to a peptide or glycopeptide.
[0133] The term "agent" or "bioactive agent" is meant to refer to
any chemical or biologic material or compound that is suitable for
delivery that induces a desired effect in or on an organism, such
as a biological or pharmacological effect, which may include, but
is not limited to a prophylactic effect, alleviating a condition
caused by a disease or a disorder, reducing or eliminating a
disease or disorder. An agent or a bioactive agent refers to
substances that are capable of exerting a biological effect in
vitro and/or in vivo. Examples include diagnostic agents,
pharmaceuticals, drugs, synthetic organic molecules, proteins,
peptides, vitamins, steroids, genetic material including
nucleotides, nucleosides, polynucleotides, RNAs, siRNAs, shRNAs,
anti-sense DNA or RNA.
[0134] The term "antibody" as used herein refers to both polyclonal
and monoclonal antibody. The term can also refer to single chain
antibodies. The term encompasses not only intact immunoglobulin
molecules, but fragments and genetically engineered derivatives of
immunoglobulin molecules as may be prepared by techniques known in
the art, and which retains the binding specificity of the antigen
binding site.
[0135] The term "fragment" refers to an amino acid segment of a
beta (1,4)-galactosyltransferase I enzyme which is able to catalyze
the linkage of a donor to an acceptor. A fragment may be from any
mammalian beta (1,4)-galactosyltransferase I. In certain
embodiments, the catalytic domain is from bovine beta
(1,4)-galactosyltransferase I, in other certain embodiments, the
catalytic domain is from human beta (1,4)-galactosyltransferase I.
In preferred embodiments, a beta (1,4)-galactosyltransferase I
fragment is encoded by SEQ ID NO: 1 or has the amino acid sequence
of SEQ ID NO: 2.
[0136] The term "donor" refers to a molecule that is actively
linked to an acceptor molecule through the action of a catalytic
domain of a galactosyltransferase, or mutant thereof. A donor
molecule can include a sugar, or a sugar derivative. Examples of
donors include, but are not limited to, UDP-GalNAc, UDP-GalNAc
analogues, UDP-galactose or UDP-galactose analogues. Donors include
sugar derivatives that include agents, biological agents, or active
groups. Accordingly, oligosaccharides may be prepared according to
the methods of the invention that include a sugar derivative having
any desired characteristic.
[0137] The term "effective amount" is meant to refer to a
sufficient amount capable to provide the desired local or systemic
effect.
[0138] The term "expression cassette" as used herein refers to a
DNA sequence capable of directing expression of a particular
nucleotide sequence in an appropriate host cell, comprising a
promoter operably linked to the nucleotide sequence of interest
that is operably linked to termination signals. It also typically
comprises sequences required for proper translation of the
nucleotide sequence. The expression cassette may be one that is
naturally occurring but has been obtained in a recombinant form
useful for heterologous expression. The expression of the
nucleotide sequence in the expression cassette may be under the
control of a constitutive promoter or of an inducible promoter that
initiates transcription only when the host cell is exposed to some
particular external stimulus. In the case of a multicellular
organism, the promoter can also be specific to a particular tissue
or organ or stage of development.
[0139] The term "beta-1,4 galactosyltransferase (beta 4Gal-T1)" as
used herein refers to enzymes substantially homologous to, and
having substantially the same biological activity as, the enzyme
encoded by the nucleotide sequence depicted in SEQ ID NO: 1 and the
amino acid sequence depicted in SEQ ID NO: 2. This definition is
intended to encompass natural allelic variations in the beta
4Gal-T1 sequence, and all references to beta 4Gal-T1, and
nucleotide and amino acid sequences thereof are intended to
encompass such allelic variations, both naturally-occurring and
man-made. The production of proteins such as the enzyme beta
4Gal-T1 from cloned genes by genetic engineering is well known.
[0140] The beta 4Gal-T1 enzyme may be synthesized in host cells
transformed with vectors containing DNA encoding the beta 4Gal-T1
enzyme. A vector is a replicable DNA construct. Vectors are used
herein either to amplify DNA encoding the beta 4Gal-T1 enzyme
and/or to express DNA which encodes the beta 4Gal-T1 enzyme. An
expression vector is a replicable DNA construct in which a DNA
sequence encoding the beta 4Gal-T1 enzyme is operably linked to
suitable control sequences capable of effecting the expression of
the beta 4Gal-T1 enzyme in a suitable host. The need for such
control sequences will vary depending upon the host selected and
the transformation method chosen. Generally, control sequences
include a transcriptional promoter, an optional operator sequence
to control transcription, a sequence encoding suitable mRNA
ribosomal binding sites, and sequences which control the
termination of transcription and translation. Amplification vectors
do not require expression control domains. All that is needed is
the ability to replicate in a host, usually conferred by an origin
of replication, and a selection gene to facilitate recognition of
transformants.
[0141] The term "immunogenic" compound or composition as used
herein refers to a compound or composition that is capable of
stimulating production of a specific immunological response when
administered to a suitable host, usually a mammal.
[0142] The term "nucleic acid" is intended to include nucleic acid
molecules, e.g., polynucleotides which include an open reading
frame encoding a polypeptide, and can further include non-coding
regulatory sequences, and introns. In addition, the terms are
intended to include one or more genes that map to a functional
locus. In addition, the terms are intended to include a specific
gene for a selected purpose. The gene can be endogenous to the host
cell or can be recombinantly introduced into the host cell, e.g.,
as a plasmid maintained episomally or a plasmid (or fragment
thereof) that is stably integrated into the genome. In one
embodiment, the gene of polynucleotide segment is involved sugar
transfer. A mutant nucleic acid molecule is intended to include a
nucleic acid molecule or gene having a nucleotide sequence which
includes at least one alteration (e.g., substitution, insertion,
deletion) such that the polypeptide or polypeptide that can be
encoded by said mutant exhibits an activity that differs from the
polypeptide or polypeptide encoded by the wild-type nucleic acid
molecule or gene.
[0143] The terms "oligosaccharide" and "polysaccharide" are used
interchangeably herein. These terms refer to saccharide chains
having two or more linked sugars. Oligosaccharides and
polysaccharides may be homopolymers and heteropolymers having a
random sugar sequence or a preselected sugar sequence.
Additionally, oligosaccharides and polysaccharides may contain
sugars that are normally found in nature, derivatives of sugars,
and mixed polymers thereof. "saccharide" refers to any of a series
of compounds of carbon, hydrogen, and oxygen in which the atoms of
the latter two elements are in the ratio of 2:1, especially those
containing the group C.sub.6H.sub.100.sub.5, including fructose,
glucose, sucrose, lactose, maltose, galactose and arabinose.
[0144] The terms "polypeptides" and "proteins" are used
interchangeably herein. Polypeptides and proteins can be expressed
in vivo through use of prokaryotic or eukaryotic expression
systems. Many such expressions systems are known in the art and are
commercially available. (Clontech, Palo Alto, Calif.; Stratagene,
La Jolla, Calif.). Examples of such systems include, but are not
limited to, the T7-expression system in prokaryotes and the
bacculovirus expression system in eukaryotes. Polypeptides can also
be synthesized in vitro, e.g., by the solid phase peptide synthetic
method or by in vitro transcription/translation systems. Such
methods are described, for example, in U.S. Pat. Nos. 5,595,887;
5,116,750; 5,168,049 and 5,053,133; Olson et al., Peptides, 9, 301,
307 ((988). The solid phase peptide synthetic method is an
established and widely used method, which is described in the
following references: Stewart et al., Solid Phase Peptide
Synthesis, W, H. Freeman Co., San Francisco (1969); Merrifield, J.
Am. Chem. Soc., 85 2149 (1963); Meienhofer in "Hormonal Proteins
and Peptides," ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp.
48-2.67; Bavaay and Merrifield, "The Peptides," eds. E. Gross and
F. Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285; and
Clark-Lewis et al., Meth. Enzymol., 287, 233 (1997). These
polypeptides can be further purified by fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reverse phase HPLC; chromatography on silica or on an
anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; or ligand affinity chromatography. The term an
"isolated polypeptide" (e.g., an isolated or purified biosynthetic
enzyme) is substantially free of cellular material or other
contaminating polypeptides from the microorganism from which the
polypeptide is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized.
[0145] The polypeptides of the invention include polypeptides
having amino acid exchanges, i.e., variant polypeptides, so long as
the polypeptide variant is biologically active. The variant
polypeptides include the exchange of at least one amino acid
residue in the polypeptide for another amino acid residue,
including exchanges that utilize the D rather than L form, as well
as other well known amino acid analogs, e.g., N-alkyl amino acids,
lactic acid, and the like. These analogs include phosphoserine,
phosphothreonine, phosphotyrosine, hydroxyproline,
gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic
acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,
penicillamine, ornithine, citruline, N-methyl-alanine,
para-benzoyl-phenylalanine, phenylglycine, propargylglycine,
sarcosine, N-acetylserine, N-formylmethionine, 3-methylhistidine,
5-hydroxylysine, and other similar amino acids and imino acids and
tert-butylglycine.
[0146] Conservative amino acid exchanges are preferred and include,
for example; aspartic-glutamic as acidic amino acids;
lysine/arginine/histidine as basic amino acids; leucine/isoleucine,
methionine/valine, alanine/valine as hydrophobic amino acids;
serine/glycine/alanine/threonine as hydrophilic amino acids.
Conservative amino acid exchange also includes groupings based on
side chains. Members in each group can be exchanged with another.
For example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine. These may be
exchanged with one another. A group of amino acids having
aliphatic-hydroxyl side chains is serine and threonine. A group of
amino acids having amide-containing side chains is asparagine and
glutamine. A group of amino acids having aromatic side chains is
phenylalanine, tyrosine, and tryptophan. A group of amino acids
having basic side chains is lysine, arginine, and histidine. A
group of amino acids having sulfur-containing side chains is
cysteine and methionine. For example, replacement of a leucine with
an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid may be accomplished to produce a
variant polypeptide of the invention.
[0147] The term "subject" as used herein refers to any animal,
including mammals, preferably humans, to which the present
invention may be applied.
[0148] The term "cancer" or "tumor" refers to an aggregate of
abnormal cells and/or tissue which may be associated with diseased
states that are characterized by uncontrolled cell proliferation.
The disease states may involve a variety, of cell types, including,
for example, endothelial, epithelial and myocardial cells. Included
among the disease states are neoplasms, cancer, leukemia and
restenosis injuries.
Beta 1,4 galactosyltransferase
[0149] Specific glycosyltransferases synthesize oligosaccharides by
the sequential transfer of the monosaccharide moiety of an
activated sugar donor to an acceptor molecule. Members of the
glycosyltransferase superfamily, which are often named after the
sugar moiety that they transfer, are divided into subfamilies on
the basis of linkage that is generated between the donor and
acceptor. Transfer of the sugar residue occurs with either the
retention (by retaining glycosyltransferases) or the inversion (by
inverting glycosyltransferases) of the configuration at the
anomeric C1 atom.
[0150] beta-1,4-Galactosyltransferases (beta4Gal-T) are a Golgi
resident, type II membrane-bound family of enzymes (beta4Gal-T1-T7)
that transfer galactose (Gal) in the presence of manganese ion
(Mn2+), from UDP-Gal to N-acetylglucosamine (GlcNAc), either free
or hound to an oligosaccharide of a glycoprotein or a glycolipid
(Brew et al., 1968; Takase et al., 1984; Powell et al, 1976; Hill,
UCLA Forum Med. Sci., 21: 63-86, 1979). This reaction allows
galactose to be linked to an N-acetylglucosamine that may itself be
linked to a variety of other molecules. Examples of these molecules
include other sugars and proteins. The reaction can be used to make
many types of molecules of biological significance. For example,
galactose-beta (1,4)-N-acetylglucosamine linkages are important for
many recognition events that control how cells interact with each
other in the body, and how cells interact with pathogens. In
addition, numerous other linkages of this type are important for
cellular recognition and binding events as well as cellular
interactions with pathogens, such as viruses.
[0151] Sequences of beta galactosyltransferase I family members
from human and other species are known, and family members exhibit
a high level of sequence identity in their catalytic domains (Lo et
al., 1998; Amado et al., 1998). DNA clones are available from
commercial resources, for example, Open Biosources.
[0152] Glycosyltransferases show great structural similarity. They
are all globular proteins with two types of fold, termed GT-A and
GT-B, which each have an N-terminal and a C-terminal domain. The
enzymes of the GT-A fold have two dissimilar domains. The
N-terminal domain, which recognizes the sugar-nucleotide donor,
comprises several b-strands that are each flanked by alpha-helices
as in a Rossmann-like fold, whereas the C-terminal domain, which
contains the acceptor-binding site, consists largely of mixed
b-sheets. By contrast, enzymes with the GT-B fold contain two
similar Rossmann-like folds, with the N-terminal domain providing
the acceptor-binding site and the C-terminal domain providing the
donor-binding site. In both types of enzyme, the two domains are
connected by a linker region and the active site is located between
the two domains. A metal-binding site is also located in the cleft
in enzymes of both the GT-B and GT-A fold (Qasba et al., 2005).
[0153] The methods of the invention are amenable to use with any
beta 1,4 galactosyltransferase 1. By any beta 1,4
galactosyltransferase I is meant from any species, for example, but
not limited to, human, bovine, or mouse. Although they have the
same donor sugar specificity, many of these are expected to
transfer Gal to different oligosaccharides containing GlcNAc at
their nonreducing end Although they have the same donor sugar
specificity, many of these are expected to transfer Gal to
different oligosaccharides containing GlcNAc at their nonreducing
end. Recent crystallographic studies on beta4Gal-T1 have provided
detailed information about the structure and function of the enzyme
(Gastinel et al., 1999; Ramakrishnan et al., 2001; Ramakrishnan et
al., 2001a; Ramakrishnan et al., 2002; Ramakrishnan et al., 2002a;
Ramakrishnan et al., 2003).
[0154] Structural studies on the beta-1,4-galactosyltransferase-1
(beta 4Gal-T1) (Ramikrishnan et al, 2004a) and on other
glycosyltransferases (Qasba et al, 2005) have shown that, upon
binding the sugar-nucleotide donor substrate, flexible loops at the
substrate binding site of these enzymes undergo a marked
conformational change, from an open to a closed conformation (Qasba
et al. 2005). This change creates an oligosaccharide
acceptor-binding site in the enzyme that did not exist before. The
loop then acts as a lid covering the bound donor substrate. After
the transfer of the glycosyl unit to the acceptor, the saccharide
product is ejected, and the loop reverts to its native conformation
to release the remaining nucleotide moiety. This conformational
change in beta 4Gal-T1 also creates the binding site for
beta-lactalbumin, a protein produced in the mammary glands during
lactation. The interaction of beta-lactalbumin with beta 4Gal-T1
changes the acceptor specificity of the enzyme from
N-acetylglucosamine (GlcNAc) to glucose (Glc), which produces
lactose that is secreted in milk. The conformational changes of
these two loops are highly coordinated. Trp314 in the small loop
plays a crucial role in the conformational state of the long loop,
in the binding of the substrates, and in the catalytic mechanism of
the enzyme (Ramakrishnan et al, 2001; Gunasekaran et al., 2003). In
the unbound state (open conformation), the side chain of Trp is
exposed to the solvent (Gastinel et al, 1999; Ramasamy et al.
2003), and the conformation of the long loop is such that the
UDP-Gal and the metal binding sites are exposed. Once the substrate
binds, the side chain of Trp314 moves into the catalytic pocket to
lock the sugar, nucleotide in its binding site. Simultaneously, the
long loop changes to its closed conformation, masking the sugar
nucleotide binding site (Ramakrishnan et al, 2001; Ramakrishnan et
al, 2003; Ramasamy et al., 2003). Furthermore, this conformational
change in the long flexible loop repositions the amino acid
residues at the N-terminal region, creating a metal ion binding
site, and at the C-terminal region, creating an
oligosaccharide-binding cavity that is also a protein-protein
interaction site for R-lactalbumin (LA) (Gasteinel et al., 1999;
Ramakrishnan et al, 2001; Ramakrishnan et al, 2003). LA is a
mammary gland-specific protein that modulates the acceptor
specificity of the enzyme toward glucose (Brodbeck et al., 1967).
LA hinds at the extended sugar binding site, present only in the
closed conformer of beta 4Gal-T1, leaving the monosaccharide
binding site of the enzyme available for the binding of Glc or
GlcNAc. Since LA competes with the oligosaccharide for binding to
the extended sugar binding site (Bell et al, 1976; Powell et al.,
1976), it is not possible to crystallize beta 4Gal-T1 in the
presence of LA with a bound oligosaccharide acceptor. The wild-type
enzyme also does not crystallize in the presence of UDP or
UDPhexanolamine, Mn2+, and oligosaccharides, thereby restricting
our structural or biochemical studies on the interactions of
oligosaccharides with beta 4Gal-T1. It has previously been shown
that the sugar moiety of the sugar nucleotide is essential for
efficiently inducing a conformational change in beta 4Gal-T1 (Geren
et al., 1975).
[0155] The reaction catalyzed by these enzymes follows a kinetic
mechanism in which the metal ion and sugar nucleotide bind to the
enzyme first, followed by the acceptor. After the glycosyl moiety
of the sugar-nucleotide donor is transferred to the acceptor with
the inversion or retention of the C1 configuration, the saccharide
product is ejected. The release of the nucleotide and the metal ion
follows, which returns the enzyme to its original state for a new
round of catalysis.
[0156] X-ray crystal structures of the catalytic domain of many
glycosyltransferases, either free or bound to substrates, have been
determined recently. These studies provide a structural basis for
the ordered binding of the donor and acceptor and for the proposed
catalytic mechanism of these enzymes (Unligil, U. M. and Rini, J.
M. (2000); Berger, E. G. and Rohrer, J; Negishi, M. et al,
(2003)).
[0157] A three-residue motif, Asp-X-Asp (DXD) or Glu-X-Asp (EXD),
or its equivalent generally participates in metal ion binding in
enzymes of the GT-A fold, Enzymes of the GT-B fold such as the
microbial glycosyltransferases MurG (Hu, Y. et al. (2003)) and GtfB
(Mulichack et al, 2001), and BUT (Morera et al. 1999), do not have
a DXD motif or its equivalent, even though some, BGT for example,
require a metal ion for activity. In glycosyltransferases that
require Mn2C ion as cofactor, the metal ion is bound in an
octahedral coordination (Qasba et al. 2005). It interacts with one
or both acidic residues of the DXD or EXD motif and with two oxygen
atoms from the a-phosphate and b-phosphate of UDP. To satisfy the
octahedral geometry, the three remaining metal ion links are made
either to water molecules or to water in combination with other
residues of the protein. In several glycosyltransferases only the
first (Lobsanov, Y. D, et al. (2004)) or the second (Gastinel et
al. 1999; Ramakrishnan et al. 2001; Ramakrishnan 2002; Unligil
2000) acidic residue of the motif coordinates directly with the
metal ion. For example, in some enzymes, the first acidic residue
of the motif either interacts directly with the sugar donor or the
ribose moiety or interacts via the water molecules coordinated to
the Mn2C ion. In blood group A and B and alpha 3GT transferases, by
contrast, both aspartic acid residues of the DXD motif directly
coordinate the metal ion.
[0158] The crystal structures of several glycosyltransferases of
either the GT-A or GT-B fold show that at least one flexible loop
region has a crucial role in the catalytic mechanism of the enzyme
(Qasba et al. 2005). Although the exact location of this loop
differs among the transferases, it is invariably located in the
vicinity of the sugar nucleotide-binding site. Owing to the
flexibility of this region, the loop structure cannot be traced in
the apo form of the enzyme, which lacks bound substrate, in the
sugar-nucleotide-bound structures, the loop either is in a closed
conformation covering the bound donor substrate or is found
disordered in the vicinity of the sugar nucleotide-binding site. In
a 3GT, the C-terminal 11-residue flexible loop changes its
conformation when the sugar nucleotide donor is bound (Boix et al.,
2001).
[0159] Of the six ligands that coordinate Mn2+, three are from
bovine beta 4Gal-T1 Asp254, Met344, and His347 (Ramakrishnan et al,
2001; Ramakrishnan et al, 2003; Boeggeman et al., 2002). Residues
Met344 and His347, separated by the hinge residue Ile345, are at
the N-terminal region of the long flexible loop. The complete metal
binding site is created only after His347 has moved during the
conformational change to coordinate with the metal ion.
[0160] In addition to GlcNAc as an acceptor, the
beta-1,4-galactosyltransferase enzyme can also use other sugars,
such as N-acyl-substituted glucosamine and N-acetyl-D-mannosamine
(Berliner, L. J. et al., Mol. Cell. Biochem., 62: 37-42 (1984)).
The beta-1,4-galactosyltransferase does not have an absolute
requirement for the sugar donor UDP-Gal; it exhibits polymorphic
donor specificity, in that it also transfers glucose (Glc),
D-deoxy-Glc, arabinose, GalNAc, and GlcNAc from their UDP
derivatives (Berliner, L. J. and Robinson, R, D., Biochemistry, 21:
6340-6343 (1982); Andree, P. J. and Berliner L. J., Biochim.
Biophys. Acta, 544: 489-495 (1982); Do, K. Y. et al., J. Biol.
Chem., 270: 18477-18451 (1995); Palcic, M. M and Hindsgaul, O.,
Glycobiology, 1: 205-209 (1991); Ramakrishnan, B. et al., J. Biol.
Chem., 276: 37665-37671 (2001)). This reaction can be used to
generate many types of molecules, as described herein, which have
applications in research and medicine.
Beta 1,4-galactosyltransferase I Doubly Substituted Enzymes
[0161] As described herein, residues in the catalytic pocket
determine the sugar donor specificities. Substitution of these
residues broaden or alter the sugar donor specificities, thus
allowing structure-based design of novel glycosyltransferases. A
three-residue motif, Asp-X-Asp (DXD) or Glu-X-Asp (EXD), or its
equivalent generally participates in metal ion binding in enzymes
of the GT-A fold. Enzymes of the GT-B fold such as the microbial
glycosyltransferases MurG (Hu, Y. et al. (2003)) and GtfB
(Mulichack et al. 2001), and BOT (Morera et al. 1999), do not have
a DXD motif or its equivalent, even though some, BGT for example,
require a metal ion for activity. In glycosyltransferases that
require Mn2C ion as cofactor, the metal ion is bound in an
octahedral coordination (Qasba et al, 2005). It interacts with one
or both acidic residues of the DXD or EXD motif and with two oxygen
atoms from the a-phosphate and b-phosphate of UDP. To satisfy the
octahedral geometry, the three remaining metal ion links are made
either to water molecules or to water in combination with other
residues of the protein. In several glycosyltransferases only the
first (Lobsanov, Y. D. et al. (2004)) or the second (Gastinel et
al. 1999; Ramakrishnan et al. 2001; Ramakrishnan 2002; Unligil
2000) acidic residue of the motif coordinates directly with the
metal ion. For example, in some enzymes, the first acidic residue
of the motif either interacts directly with the sugar donor or the
ribose moiety or interacts via the water molecules coordinated to
the Mn2C ion. In blood group A and B and alpha 3GT transferases, by
contrast, both aspartic acid residues of the DXD motif directly
coordinate the metal ion.
[0162] The crystal structures of several glycosyltransferases of
either the GT-A or GT-B fold show that at least one flexible loop
region has a crucial role in the catalytic mechanism of the enzyme
(Qasba et al. 2005). Although the exact location of this loop
differs among the transferases, it is invariably located in the
vicinity of the sugar nucleotide-binding site. Owing to the
flexibility of this region, the loop structure cannot be traced in
the apo form of the enzyme, which lacks hound substrate. In the
sugar-nucleotide-bound structures, the loop either is in a closed
conformation covering the bound donor substrate or is found
disordered in the vicinity of the sugar nucleotide-binding site. In
alpha 3GT, the C-terminal 11-residue flexible loop changes its
conformation when the sugar nucleotide donor is bound (Boix et al.,
2001).
[0163] The instant invention describes catalytic domains of beta
1,4-galactosyltransferase I with amino acid exchanges.
[0164] The instant invention describes catalytic domains of beta
1,4-galactosyltransferase with amino acid substitutions or
exchanges. FIG. 6 shows the nucleotide sequence corresponding to
human beta 1,4 galactosyltransferase-I (SEQ ID NO: 5) according to
NCBI Accession No. NM.sub.--001005417, and FIG. 7 shows the amino
acid sequence corresponding to human beta 1,4
galactosyltransferase-I (SEQ ID NO: 6) according to NCBI Accession
No. NM.sub.--001005417. One of skill in the art using the guidance
provided herein can make amino acid substitutions or exchanges at
residues of beta 1,4-galactosyltransferase I that broaden or alter
donor specificity.
[0165] In certain examples, the amino acid exchange is an amino
acid at positions 285 and 340, corresponding to human beta
1,4-galactosyltransferase I. In other certain examples, the amino
acid exchange is an amino acid at positions 289 and 344
corresponding to bovine beta 1,4-galactosyltransferase I (see, for
example, PCT/US2004/000470 and US Application No. 20060084162, both
of which are incorporated herein by reference in their entireties).
The corresponding tyrosine in the mouse beta
1,4-galactosyltransferase I is located at amino acid position 286.
Other examples of specific exchanges are Y289I and Y289N,
corresponding to Y2851 and Y285N in human.
[0166] Those of skill in the art can readily determine equivalent
amino acids in other beta 1,4-galactosyltransferase I catalytic
domains and generate them through recombinant techniques known in
the art.
[0167] Included in the invention are polypeptide fragments from a
beta (1,4)-galactosyltransferase I that transfers GalNAc or
galactose from a sugar donor to a sugar acceptor in the presence of
magnesium. In certain examples, the catalytic domain comprises an
amino acid exchange at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I. In other
examples, the catalytic domain comprises a conservative amino acid
exchange at amino acid positions 289 and 344 corresponding to
bovine beta (1,4)-galactosyltransferase I.
[0168] In the human sequence of the enzyme, the Leucine (L) can be
exchanged for a tyrosine (Y) at amino acid position 285 and a
methionine (M) is exchanged for a histidine (H) at amino acid
position 340. In the bovine sequence of the enzyme, a leucine (L)
is exchanged for a tyrosine (Y) at amino acid position 289 and a
methionine (M) is exchanged for a histidine (H) at amino acid
position 344.
[0169] In preferred examples, the invention features a polypeptide
fragment from a beta (1,4)-galactosyltransferase I that transfers
GalNAc or galactose from a sugar donor to a sugar acceptor, wherein
the isolated catalytic domain comprises SEQ ID NO: 1.
[0170] In certain preferred embodiments of the invention, the
catalytic domain corresponds to amino acids 127-398 of the doubly
substituted mutant human beta-4Gal-T1-M340H-Y285L. SEQ ID NO: 1
represents the DNA sequence correspond ng to the protein sequence,
amino acid residues 127 to 398:
TABLE-US-00001 (SEQ ID NO: 1) 1 CTGCCCGCAT GCCCTGAGGA GTCCCCGCTG
CTTGTGGGCC CCATGCTGAT 51 TGAGTTTAAC ATGCCTGTGG ACCTGGAGCT
CGTGGCAAAG CAGAACCCAA 101 ATGTGAAGAT GGGCGGCCGC TATGCCCCCA
GGGACTGCGT CTCTCCTCAC 151 AAGGTGGCCA TCATCATTCC ATTCCGCAAC
CGGCAGGAGC ACCTCAAGTA 201 CTGGCTATAT TATTTGCACC CAGTCCTGCA
GCGCCAGCAG CTGGACTATG 251 GCATCTATGT TATCAACCAG GCGGGAGACA
CTATATTCAA TCGTGCTAAG 301 CTCCTCAATG TTGGCTTTCA AGAAGCCTTG
AAGGACTATG ACTACACCTG 351 CTTTGTGTTT AGTGACGTGG ACCTCATTCC
AATGAATGAC CATAATGCGT 401 ACAGGTGTTT TTCACAGCCA CGGCACATTT
CCGTTGCAAT GGATAAGTTT 451 GGATTCAGCC TACCTTATGT TCAGTTGTTT
GGAGGTGTCT CTGCTCTAAG 501 TAAACAACAG TTTCTAACCA TCAATGGATT
TCCTAATAAT TATTGGGGCT 551 GGGGAGGAGA AGATGATGAC ATTTTTAACA
GATTAGTTTT TAGAGGCATG 601 TCTATATCTC GCCCAAATGC TGTGGTCGGG
AGGACGCGTC ACATCCGCCA 651 CTCGAGAGAC AAGAAAAATG AACCCAATCC
TCAGAGGTTT GACCGAATTG 701 CACACACAAA GGAGACAATG CTCTCTAATG
GTTTGAACTC ACTCACCTAC 751 CAGGTGCTGG ATGTACAGAG ATACCCATTG
TATACCCAAA TCACAGTGGA 801 CATCGGGACA CCGAGCTAG
SEQ ID NO: 2 represents the protein sequence corresponding to amino
acid residues 127 to 398:
TABLE-US-00002 (SEQ ID NO: 2) 127 LPACPEESPL LVGPMLIEFN MPVDLELVAK
QNPNVKMGGR YAPRDCVSPH 177 KVAIIIPFRN RQEHLKYWLY YLHPVLQRQQ
LDYGIYVINQ AGDTIFNRAK 227 LLNVGFQEAL KDYDYTCFVF SDVDLIPMND
HNAYRCFSQP RHISVAMDKF 277 GFSLPYVQLF GGVSALSKQQ FLTINGFPNN
YWGWGGEDDD IFNRLVFRGM 327 SISRPNAVVG RTRHIRHSRD KKNEPNPQRF
DRIAHTKETM LSNGLNSLTY 377 QVLDVQRYPL YTQITVDIGT PS*
[0171] A number of sugar donors are available, and are selected
from UDP-GalNAc, UDP-GalNAc analogues, UDP-galactose, or
UDP-galactose analogues. The UDP-GalNAc analogues or UDP-galactose
analogue can comprise an azido group, a keto group, or a thiol
group. In order to link an agent to the UDP-galactose analogue, the
azido group, the keto group or the thiol group is substituted at
the C2 position of galactose. The invention enables a range of
agents to be linked to the sugar moiety of the sugar donor. For
instance, the agent can be selected from antibodies, single chain
antibodies, bacterial toxins, growth factors, therapeutic agents,
targeting agents, contrast agents, chemical labels, a radiolabels,
and fluorescent labels.
[0172] The sugar acceptor is preferably N-acetylglucosamine
(GlcNAc). The N-acetylglucosamine (GlcNAc) can free or attached to
a peptide of a glycopeptide.
[0173] Flexible loops undergo conformational changes upon substrate
binding and create the acceptor binding site and the catalytic
pocket. Metal ions often bind at the hinge region of the flexible
loop. Since 5 mM Mn2+ is toxic to live cells, the Mg2+ dependent
b4Gal-Y285L M340H-Gal-T1double mutant, is a better choice for the
galactosylation of live cells. The sugar transfer of the instant
invention is unique in that in preferred embodiments sugar transfer
occurs in the presence of magnesium.
Nucleic Adds and Vectors
[0174] The present invention provides isolated nucleic acid
segments that encode catalytic domains of double mutant beta 1,4
galactosyltransferase, for example in certain embodiments human
beta-1,4-Tyr285Leu-Met340His-Gal-T1. Nucleic acid sequences
encoding a double mutant beta 1,4 galactosyltransferase, human
beta-1,4-Tyr285Leu-Met340His-Gal-T1, for example SEQ ID NO: 1, as
well as other beta 1,4 galactosyltransferases from other organisms
are available. These nucleic acid sequences can be modified to
encode the polypeptide fragments and amino acid segments of the
invention through use of well-known techniques (Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (2001)). For example, a
portion of the nucleic acid sequence encoding beta 1,4
galactosyltransferase, for example SEQ ID NO: 1, can be inserted
into an expression vector such that an amino acid segment
corresponding to the catalytic domain of the double mutant beta 1,4
galactosyltransferase, human beta-1,4-Tyr285Leu-Met340His-Gal-T1
(SEQ ID NO: 2) is expressed upon transformation of a cell with the
expression vector. The nucleic acid segments of the invention may
be optimized for expression in select cells. Codon optimization
tables are available. Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1988.
[0175] The nucleic acid segments can be inserted into numerous
types of vectors. A vector may include, but is not limited to, any
plasmid, phagemid, F-factor, virus, cosmid, or phage in double or
single stranded linear or circular form, which may or may not be
self-transmissible or mobilizable. The vector can also transform a
prokaryotic or eukaryotic host either by integration into the
cellular genome or exist extrachromosomally (e.g. autonomous
replicating plasmid with an origin of replication).
[0176] Preferably the nucleic acid segment in the vector is under
the control of, and operably linked to, an appropriate promoter or
other regulatory elements for transcription in vitro or in a host
cell such as a eukaryotic cell or microbe, e.g. bacteria. The
vector may be a hi-functional expression vector which functions in
multiple hosts. In the case of genomic DNA, this may contain its
own promoter or other regulatory elements and in the case of cDNA
this may be under the control of a promoter or other regulatory
sequences for expression in a host cell.
[0177] Specifically included are shuttle vectors by which is meant
a DNA vehicle capable, naturally or by design, of replication in
two different host organisms, which may be selected from bacteria
and eukaryotic cells (e.g. mammalian, yeast or fungal).
[0178] The vector may also be a cloning vector which typically
contains one or a small number of restriction endonuclease
recognition sites at which nucleic acid segments can be inserted in
a determinable fashion. Such insertion can occur without loss of
essential biological function of the cloning vector. A cloning
vector may also contain a marker gene that is suitable for use in
the identification and selection of cells transformed with the
cloning vector. Examples of marker genes are tetracycline
resistance, hygromycin resistance or ampicillin resistance. Many
cloning vectors are commercially available (Stratagene, New England
Biolabs, Clonetech).
[0179] The nucleic acid segments of the invention may also be
inserted into an expression vector. Typically an expression vector
contains (1) prokaryotic DNA elements coding for a bacterial
replication origin and an antibiotic resistance gene to provide for
the amplification and selection of the expression vector in a
bacterial host; (2) regulatory elements that control initiation of
transcription such as a promoter; and (3) DNA elements that control
the processing of transcripts such as introns, transcription
termination/polyadenylation sequence.
[0180] Methods to introduce a nucleic acid segment into a vector
are well known in the art (Sambrook et al., 1989). Briefly, a
vector into which the nucleic acid segment is to be inserted is
treated with one or more restriction enzymes (restriction
endonuclease) to produce a linearized vector having a blunt end, a
"sticky" end with a 5' or a 3' overhang, or any combination of the
above. The vector may also be treated with a restriction enzyme and
subsequently treated with another modifying enzyme, such as a
polymerase, an exonuclease, phosphatase or a kinase, to create a
linearized vector that has characteristics useful for ligation of a
nucleic acid segment into the vector. The nucleic acid segment that
is to be inserted into the vector is treated with one or more
restriction enzymes to create a linearized segment having a blunt
end, a "sticky" end with a 5' or a 3' overhang, or any combination
of the above. The nucleic acid segment may also be treated with a
restriction enzyme and subsequently treated with another DNA
modifying enzyme. Such DNA modifying enzymes include, but are not
limited to, polymerase, exonuclease, phosphatase or a kinase, to
create a polynucleic acid segment that has characteristics useful
for ligation of a nucleic acid segment into the vector.
[0181] The treated vector and nucleic acid segment are then ligated
together to form a construct containing a nucleic acid segment
according to methods known in the art (Sambrook, 2002). Briefly,
the treated nucleic acid fragment and the treated vector are
combined in the presence of a suitable buffer and ligase. The
mixture is then incubated under appropriate conditions to allow the
ligase to ligate the nucleic acid fragment into the vector. It is
preferred that the nucleic acid fragment and the vector each have
complimentary "sticky" ends to increase ligation efficiency, as
opposed to blunt-end ligation. It is more preferred that the vector
and nucleic acid fragment are each treated with two different
restriction enzymes to produce two different complimentary "sticky"
ends. This allows for directional ligation of the nucleic acid
fragment into the vector, increases ligation efficiency and avoids
ligation of the ends of the vector to reform the vector without the
inserted nucleic acid fragment.
[0182] Suitable prokaryotic vectors include but are not limited to
pBR322, pMB9, pUC, lambda bacteriophage, m13 bacteriophage, and
Bluescript.RTM. Suitable eukaryotic vectors include but are not
limited to PMSG, pAV009/A+, PMTO10/A+, pMAM neo-5, bacculovirus,
pDSVE, YIP5, YRP17, YEP. It will be clear to one of ordinary skill
in the art which vector or promoter system should be used depending
on which cell type is used for a host cell.
[0183] The invention also provides expression cassettes which
contain a control sequence capable of directing expression of a
particular nucleic acid segment of the invention either in vitro or
in a host cell. The expression cassette is an isolatable unit such
that the expression cassette may be in linear form and functional
in in vitro transcription and translation assays. The materials and
procedures to conduct these assays are commercially available from
Promega Corp. (Madison, Wis.). For example, an in vitro transcript
may be produced by placing a nucleic acid segment under the control
of a T7 promoter and then using T7 RNA polymerase to produce an in
vitro transcript. This transcript may then be translated in vitro
through use of a rabbit reticulocyte lysate. Alternatively, the
expression cassette can be incorporated into a vector allowing for
replication and amplification of the expression cassette within a
host cell or also in vitro transcription and translation of a
nucleic acid segment.
[0184] Such an expression cassette may contain one or a plurality
of restriction sites allowing for placement of the nucleic acid
segment under the regulation of a regulatory sequence. The
expression cassette can also contain a termination signal operably
linked to the nucleic acid segment as well as regulatory sequences
required for proper translation of the nucleic acid segment.
Expression of the nucleic acid segment in the expression cassette
may be under the control of a constitutive promoter or an inducible
promoter, which initiates transcription only when the host cell is
exposed to some particular external stimulus.
[0185] The expression cassette may include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region, a nucleic acid segment and a transcriptional and
translational termination region functional in vivo and/or in
vitro. The termination region may be native with the
transcriptional initiation region, may be native with the nucleic
acid segment, or may be derived from another source. Numerous
termination regions are known in the art. Guerineau et al., Mol.
Gen. Genet., 262:141 (1991); Proudfoot, Cell, 64:671 (1991);
Sanfacon et al., Genes Dev., 5:141 (1991); Munroe et al., Gene,
91:151 (1990); Ballas et al., Nucleic Acids Res., 17:7891 (1989);
Joshi et al., Nucleic Acid Res., 15:9627 (1987).
[0186] The regulatory sequence can be a nucleic acid sequence
located upstream (5' non-coding sequences), within, or downstream
(3' non-coding sequences) of a coding sequence, and which
influences the transcription, RNA processing or stability, or
translation of the associated coding sequence. Regulatory sequences
can include, but are not limited to, enhancers, promoter and
repressor binding sites, translation leader sequences, introns, and
polyadenylation signal sequences. They may include natural and
synthetic sequences as well as sequences that may be a combination
of synthetic and natural sequences. While regulatory sequences are
not limited to promoters, some useful regulatory sequences include
constitutive promoters, inducible promoters, regulated promoters,
tissue-specific promoters, viral promoters and synthetic
promoters.
[0187] A promoter is a nucleotide sequence that controls expression
of the coding sequence by providing the recognition for RNA
polymerase and other factors required for proper transcription. A
promoter includes a minimal promoter, consisting only of all basal
elements needed for transcription initiation, such as a TATA-box
and/or initiator that is a short DNA sequence comprised of a
TATA-box and other sequences that serve to specify the site of
transcription initiation, to which regulatory elements are added
for control of expression. A promoter may be inducible. Several
inducible promoters have been reported (Current Opinion in
Biotechnology, 7:168 (1996)). Examples include the tetracycline
repressor system, Lac repressor system, copper-inducible systems,
salicylate-inducible systems (such as the PR1a system). Also
included are the benzene sulphonamide (U.S. Pat. No. 5,364,780,
incorporated by reference herein) and alcohol- (WO 97/06269 and WO
97/06268, both incorporated by reference herein) inducible systems
and glutathione S-transferase promoters. In the case of a
multicellular organism, the promoter can also be specific to a
particular tissue or organ or stage of development.
[0188] An enhancer is a DNA sequence which can stimulate promoter
activity and may be an innate element of the promoter or a
heterologous element inserted to enhance the level or tissue
specificity of a promoter. It is capable of operating in both
orientations (normal or flipped), and is capable of functioning
even when moved either upstream or downstream from the promoter.
Both enhancers and other upstream promoter elements bind
sequence-specific DNA-binding proteins that mediate their
effects.
[0189] The expression cassette can contain a 5' non-coding sequence
which is a nucleotide sequence located 5' (upstream) to the coding
sequence. It is present in the fully processed mRNA upstream of the
initiation codon and may affect processing of the primary
transcript to mRNA, stability of the mRNA, or translation
efficiency (Turner et al., Molecular Biotechnology, 3:225
(1995)).
[0190] The expression cassette may also contain a 3' non-coding
sequence, which is a nucleotide sequence, located 3' (downstream)
to a coding sequence and includes polyadenylation signal sequences
and other sequences encoding regulatory signals capable of
affecting mRNA processing or gene expression. The polyadenylation
signal is usually characterized by affecting the addition of
polyadenylic acid tracts to the 3' end of the mRNA precursor.
[0191] The invention also provides a construct containing a vector
and an expression cassette. The vector may be selected from, but
not limited to, any vector previously described. Into this vector
may be inserted an expression cassette through methods known in the
art and previously described (Sambrook et al, 1989). In one
embodiment, the regulatory sequences of the expression cassette may
be derived from a source other than the vector into which the
expression cassette is inserted. In another embodiment, a construct
containing a vector and an expression cassette is formed upon
insertion of a nucleic acid segment of the invention into a vector
that itself contains regulatory sequences. Thus, an expression
cassette is formed upon insertion of the nucleic acid segment into
the vector. Vectors containing regulatory sequences are available
commercially and methods for their use are known in the art
(Clonetech, Promega, Stratagene).
[0192] The expression cassette, or a vector construct containing
the expression cassette may be inserted into a cell. The expression
cassette or vector construct may be carried episomal or integrated
into the genome of the cell.
[0193] A variety of techniques are available and known to those
skilled in the art for introduction of constructs into a cellular
host. Transformation of bacteria and many eukaryotic cells may be
accomplished through use of polyethylene glycol, calcium chloride,
viral infection, phage infection, electroporation and other methods
known in the art. Other transformation methods are available to
those skilled in the art, such as direct uptake of foreign DNA
constructs (see EP 295959, incorporated by reference herein),
techniques of electroporation or high velocity ballistic
bombardment with metal particles coated with the nucleic acid
constructs (U.S. Pat. No. 4,945,050, incorporated by reference
herein).
[0194] The selection of an appropriate expression vector will
depend upon the method of introducing the expression vector into
host cells. Typically an expression vector contains (I) prokaryotic
DNA elements coding for a bacterial origin of replication and an
antibiotic resistance gene to provide for the amplification and
selection of the expression vector in a bacterial host; (2) DNA
elements that control initiation of transcription, such as a
promoter; (3) DNA elements that control the processing of
transcripts, such as introns, transcription
termination/polyadenylation sequence; and (4) a reporter gene that
is operatively linked to the DNA elements to control transcription
initiation. Useful reporter genes include beta-galactosidase,
chloramphenicol acetyl transferase, luciferase, green fluorescent
protein (GFP) and the like.
Methods of Making and Folding
[0195] Galactosyltransferase enzymes of the invention may be
produced in soluble form. Methods that may be used to produce such
soluble enzymes have been described (U.S. Pat. No. 5,032,519,
incorporated by reference in its entirety herein). Briefly, a
hydrophobic transmembrane anchor region of a galactosyltransferase
is removed to produce an enzyme that is in soluble form.
[0196] Alternatively, 1,4 beta galactosyltransferase enzymes of the
invention may be produced such that they are anchored in the
membrane of a cell. Such enzymes may be produced that are anchored
in the membranes of prokaryotic and eukaryotic cells. Methods to
produce such enzymes have been described (U.S. Pat. No. 6,284,493,
incorporated by reference in its entirety herein).
[0197] Briefly, in the case of procaryotes, the signal and
transmembrane sequences of the transferase, for example the mutant
1,4 beta galactosyltransferase of the invention, are replaced by a
bacterial signal sequence, capable of effecting localization of the
fusion protein to the outer membrane. Suitable signal sequences
include, but are not limited to those from the major E. coli
lipoprotein Lpp and lam B. In addition, membrane spanning regions
from Omp A, Omp C, Omp F or Pho E can be used in a tripartite
fusion protein to direct proper insertion of the fusion protein
into the outer membrane. Any prokaryotic cells can be used in
accordance with the present invention including but not limited to
E. coli, Bacillus sp., and Pseudomonas sp. as representative
examples.
[0198] It is also possible, in certain embodiments, that the native
transmembrane domain of the glycosyltransferase, for example the
engineered 1,4 beta galactosyltransferase of the invention as
described herein, is replaced by the transmembrane domain of a
bacterial outer membrane protein. For example, the 1,4 beta
galactosyltransferase signal sequence and the bacterial
transmembrane region act in concert to anchor the
galactosyltransferase to the bacterial outer cell membrane. Nearly
any outer membrane bound protein is suitable for this use including
but not limited to Omp A, Omp C, and Omp F, Lpp, and Lam R. The
catalytic portion of the 1,4 beta galactosyltransferase should be
fused to an extracellular loop in the bacterial transmembrane
region in order to insure proper orientation of the fusion protein
on the outer membrane surface and not in the cytoplasm or periplasm
of the cell. Insertion of a protein into such a loop region has
been previously reported (Charbit et al., J. Bacteriology, 173:262
(1991); Francisco et al., Proc. Natl. Acad. Sci.,
89:2713(1992)).
[0199] The present invention is also applicable for use with
eukaryotic cells resulting in cell surface expression of
glycosyltransferases in known culturable eukaryotic cells including
but not limited to yeast cells, insect cells, chinese hamster ovary
cells (CHO cells), mouse L cells, mouse A9 cells, baby hamster
kidney cells, C127 cells, COS cells, Sf9 cells, and PC8 cells.
[0200] In another example of the present invention, the
transmembrane domain of the glycosyltransferase is replaced by the
transmembrane domain of a plasma membrane protein. The
transmembrane domain of any resident plasma membrane protein will
be appropriate for this purpose. For example, but not to be
limiting, the transmembrane portions of the M6 P/IGF-II receptor,
LDL receptor or the transferrin receptor are representative
examples.
[0201] In another embodiment the Golgi retention signal of the
glycosyltransferase is disrupted by site-directed mutagenesis. This
approach mutates the amino acids responsible for localizing the
galactosyltransferase to the Golgi compartment. The resultant
glycosyltransferase is transported to the plasma membrane where it
becomes anchored via its modified transmembrane sequences.
[0202] In vitro folding of 1,4 beta galactosyltransferase requires
proper disulfide bond formation. Ways to ensure proper disulfide
bond formation include S-sulfonation of the protein prior to
disulfide formation, use of oxido-shuffling reagents, and mutation
of free Cys residue to Thr. In the in vitro folding of beta4GalT1,
the stem region acts as a chaperone. Additionally, there are
additives that can be used to prevent the hydrophobic collapse,
including polyethylene glycol (PEG, e.g. PEG-4000) or
L-arginine-HCl. PEG-4000 and L-arginine are thought to beneficially
affect the solubility of folding intermediates of both catalytic
domain-proteins (CD-proteins) and stem region/catalytic domain
proteins (SRCD-proteins) during in vitro folding or protein
obtained from inclusion bodies. In the case of catalytic domain
(CD)-proteins, the majority of misfolded proteins are insoluble in
the absence of PEG-4000 and L-arginine and so they precipitate out
during dialysis. Thus, the process will leave behind the properly
folded molecules in solution bound to UDP-agarose that are
enzymatically active.
[0203] Beta (1,4)-galactosyltransferase I is a type II Golgi
resident protein with a short cytoplasmic tail, a transmembrane
domain followed by a stem region and has a globular catalytic
domain that faces the Golgi lumen. When the catalytic domain of
beta (1,4)-galactosyltransferase I is expressed in E. Coli, it
forms insoluble inclusion bodies. General methods for isolating and
folding inclusion bodies containing galactosyltransferase catalytic
domains have been previously described (Ramakrishnan et al., J.
Biol. Chem., 276:37665 (2001)). These inclusion bodies can be
collected and then solubilized and folded in vitro to produce
catalytically active domains. Thus, the in vitro folding efficiency
is directly related to the quantity of active enzyme that is
produced from the isolated inclusion bodies. Accordingly, methods
to increase the in vitro folding efficiency would provide increased
production of catalytic domains that can be used to create useful
products. US Application 20060084162, incorporated by reference in
its entirety herein, provides materials and methods that improve in
vitro folding of catalytic domains from galactosyltransferases that
are related to the use of a stem region of beta
(1,4)-galactosyltransferase I. Such methods are of use in the
instant invention.
Methods of the Invention
[0204] The methods as described herein provide the ability to
conjugate multiple agents to compounds or compositions of the
invention. An embodiment of the present invention provides a
glycoconjugate in which one or more bioactive agents are bound to a
modified saccharide (e.g. a sugar) residue, for example, a modified
galactose, which is in turn bound to a targeting compound, e.g., a
compound capable of binding a receptor on a cell membrane. In this
manner, many targeting glycoconjugates can be constructed. An
example, not meant to be limiting, is a gene delivery system for
genetic therapy that can be produced by binding a nucleotide and a
ligand or antibody to the modified sugar. A therapeutic compound
for cancer can be produced by binding a chemotherapeutic agent and
a ligand or antibody, e.g., an antibody to a cancer antigen, to the
modified sugar residue.
[0205] The glycoconjugates can be manufactured as designer
glycoconjugates, according to therapeutic need. As such, the
designer polypeptide itself can be used for the targeting and drug
delivery. The glycoconjugates can be manufactured as nanoparticles.
In certain examples, a biological substrate, such as a bioactive
agent, for example a therapeutic agent, is used to engineer the
nanoparticle. In other examples a second, third, fourth or more
bioactive polypeptide is used in association with the nanoparticle
to engineer multivalent nanoparticles. The bioactive agents do not
have to be the same, for example a nanoparticle comprising three
bioactive agents may comprise a chemotherapeutic, a tracking agent
and a targeted delivery agent, such as an antibody.
[0206] The glycoconjugates can be manufactured according to the
methods described herein can be nanoparticles. Nanoparticles of the
invention have use in methods of treating diseases.
[0207] In other examples, the methods of the invention are used to
engineer a glycoprotein from a magnetic resonance agent for use in
diagnostic therapies. In these preferred examples, nanoparticles
are engineered as described herein, where the nanoparticles are
superparamagnetic nanoparticle.
[0208] Catalytic domains of the invention having altered donor and
acceptor specificity can be used to catalyze the linkage of
numerous sugars from a donor to numerous acceptor sugars. Linkage
of sugar derivatives can also achieved through use of the altered
catalytic domains of the invention due to their expanded donor and
acceptor specificity.
[0209] The presence of modified sugar moieties on a glycoprotein
makes it possible to link bioactive molecules via modified glycan
chains, thereby assisting in the assembly of bionanoparticles that
are useful for developing the targeted drug delivery system and
contrast agents for example for use in imaging e.g., magnetic
resonance imaging. The reengineered recombinant
glycosyltransferases as described herein also make it possible to
remodel the oligosaccharide chains of glycoprotein drugs, and to
synthesize oligosaccharides for vaccine development.
[0210] Targeted Glycoconjugates
[0211] Beta 1,4-galactosyltansferase (GaIT) catalyzes the transfer
of galactose from the donor UDP-galactose, to an acceptor,
N-acetylglucosamine (GlcNAc, present at the non-reducing terminal
end of glycans of glycoproteins and glycolipids, to form a
galactose-beta-1,4-N-acetylglucosamine bond (Hill, UCLA Forum Med.
Sci., 21: 63-86 (1979). This reaction allows galactose to be linked
to an N-acetylglucosamine that may itself be linked to a variety of
other molecules, such as sugars and proteins, e.g., antibodies. In
addition to GlcNAc as an acceptor, the enzyme can also use other
sugars, such as N-acyl-substituted glucosamine and
N-acetyl-D-mannosamine (Berliner, L. J. et al., Mol. Cell.
Biochem., 62: 37-42 (1984)). The enzyme does not have an absolute
requirement for the sugar donor UDP-Gal; instead, it exhibits
polymorphic donor specificity, in that it also transfers glucose
(Glc), D-deoxy-Glc, arabinose, GaINAc, and GlcNAc from their UDP
derivatives (Berliner, L. J. and Robinson, R. D., Biochemistry, 21:
6340-6343 (1982); Andree, P. J. and Berliner L. J., Biochim.
Biophys. Acta, 544: 489-495 (1982); Do, K. Y. et al., J. Biol.
Chem., 270: 18477-1845(1995); Palcic, M. M and Hindsgaul, O.,
Glycobiology, 1: 205-209 (1991); Ramakrishnan, B. et al., J. Biol.
Chem., 276: 37665-37671 (2001)). This reaction can be used to
generate many types of molecules, as described herein, which have
applications in research and medicine.
[0212] As described herein, modifications in sugar donors, for
example UDP-GalNAc analogues or UDP-galactose analogues, are
tolerated by the beta 1,4 galactosyltransferase mutants. The beta
1,4 galT mutants of the invention have the ability to use unnatural
substrates, due to altered donor specificity, in sugar transfer
reactions. For example, in one embodiment, the catalytic domain of
GalT has a tyrosine exchanged with another amino acid at an amino
acid position corresponding to 289 in the bovine beta
(1,4)-galactosyltransferase I (see, for example, PCT/US2004/000470,
filed Jan. 9, 2004, which is incorporated herein by reference) and
a tyrosine exchanged with another amino acid at an amino acid
position corresponding to 285 in the human beta
(1,4)-galactosyltransferase I.
[0213] One of skill in the art can readily determine equivalent
amino acids in other (1,4)-galactosyltransferase I catalytic
domains and generate amino acid exchanges through recombinant
techniques known in the art. In one embodiment, a genetically
engineered form of beta (1,4)-galactosyltransferase I that
transfers GalNAc or galactose from a sugar donor to a sugar
acceptor glycol-polypeptide in the presence of magnesium is used to
catalyze the formation of the glycoconjugates of the invention.
This genetically engineered form of beta 1,4 Gaff has an enlarged
binding pocket which enhances the catalytic activity toward GalNAc
substrates without compromising specificity (See, Khidekel et al.,
2003 and PCT/US04/00470, filed Jan. 9.2004, both of which are
incorporated herein by reference).
[0214] In one embodiment of the invention, the donor sugar is
modified so as to include a functional group at the C2 position of
the sugar ring, preferably a ketone or an azido or a thiol
functionality. In another embodiment, the modified sugar is a
galactose or a GalNAc analogue, which is modified at the C2
position by the addition of ketone functionality.
[0215] WO 2005/051429, incorporated by reference in its entirety
herein, describes methods used to bind a bioactive agent to the
modified sugar. The bioactive compounds may preferably include a
functional group which may be useful, for example, in forming
covalent bonds with the sugar residue, which are not generally
critical for the activity of the bioactive agent. Examples of such
functional groups include, for example, amino(--NH:2),
hydroxy(--OH), carboxyl (--COOH), thiol (--SH), phosphate,
phosphinate, ketone group, sulfate and sulfinate groups. If the
bioactive compounds do not contain a useful group, one can be added
to the bioactive compound by, for example, chemical synthetic
means. Where necessary and/or desired, certain moieties on the
components may be protected using blocking groups, as is known in
the art, see, e.g., Green & Wuts, Protective Groups in Organic
Synthesis (John Wiley & Sons)(1991).
[0216] Exemplary covalent bonds by which the bioactive compounds
may be associated with the sugar residue include, for example,
amide (--CONH--); thioamide (--CSNH--); ether (ROR', where R and R'
may be the same or different and are other than hydrogen); ester
(--COO--); thioester (--COS--); -0-; --S--; --Sn--, where n is
greater than 1, preferably about 2 to about 8; carbamates; --NH--;
--NR--, where R is alkyl, for example, alkyl of from about 1 to
about 4 carbons; urethane; and substituted imidate; and
combinations of two or more of these.
[0217] Covalent bonds between a bioactive agent and a modified
sugar residue may be achieved through the use of molecules that may
act, for example, as spacers to increase the conformational and
topographical flexibility of the compound. Examples of such spacers
include, for example, succinic acid, 1,6-hexanedioic acid,
1,8-octanedioic acid, and the like, as well as modified amino
acids, such as, for example, 6-aminohexanoic acid, 4-aminobutanoic
acid, and the like.
[0218] One of skill in the art can easily chose suitable compatible
reactive groups for the bioactive agent and the modified sugar, so
as to generate a covalent bond between the bioactive agent and the
modified sugar. Also, while the glycoconjugates of the invention
are generally described with the targeting agent as the acceptor
molecule or structure onto which a donor molecule (e.g.,
UDP-galactose) is actively linked through the action of a catalytic
domain of a galactosyltransferase, or mutant thereof, the bioactive
agent can also be an acceptor molecule.
[0219] In certain embodiments, the instant method can be used to
monitor glycosylation, for example the glycosylation of therapeutic
glycoproteins and monoclonal antibodies. The potential of
glycosyltransferase mutants to produce glycoconjugates carrying
sugar moieties with reactive groups may be a benefit to the
glycotargeting of drugs to their site of action. Although a great
number of pharmaceutical agents are discovered each year, the
clinical application of these is many times hindered because of
failure to reach the site of action. The methods described herein
that include using mutant glycosyltransferases to transfer
chemically reactive sugar residues for linking of other molecules
via specific glycan chains may be used as an efficient drug
delivery system.
[0220] Detection
[0221] The beta (1,4) glycosyltransferases as described herein have
application in the detection of specific sugar residues on a glycan
chain of a glycoconjugates and in the glycoconjugation and assembly
of bio-nanoparticles for the targeted delivery of bioactive agents.
Protein glycoslation is one of the most abundant posttranslational
modifications and plays a fundamental role in the control of
biological systems and in disease. The O-GlcNAc modification on
proteins is the dynamic posttranslational modification in which the
beta-N-acetylglucosamine is covalently attached to serine or
threonine residues in proteins.
[0222] Accordingly, glycosylation has been found to be a marker in
disease. For example, in cancer, tumor
beta-1,4-galactosyltransferase IV overexpression is closely
associated with colorectal cancer metastasis and poor prognosis.
Chen W S et al. Clin Cancer Res. 2005 Dec. 15; 11(24 Pt
1):8615-22). Further, carbohydrate modifications have been shown to
be important for host-pathogen interactions, inflammation,
development, and malignancy (Varki, 1993; Lasky, 1996).
[0223] Several methods have been reported for the identification of
O-GlcNAc modification on proteins. One of the detection methods
involves the enzymatic labeling by beta 4Gal-T1 of G-GlcNAc using
UDP-3H galactose; however, this method is time-consuming and
expensive.
[0224] The methods described herein offer the advantages the
modification occurs in a site directed manner, only where the
carbohydrate is attached to the glycoprotein. Such specificity
permits, for example, the use of site-directed immunotherapy
without affecting the antigen binding affinity of the
immunoglobulin. Such specificity permits, further, the potential
use of this approach in developing a drug delivery system or
biological probes.
[0225] Imaging
[0226] Included in the invention are methods for imaging a target
cell or tissue in a subject. The methods as described herein
comprise administering to a subject a polypeptide fragment
synthesized by the method comprising incubating a reaction mixture
comprising a polypeptide fragment from a beta
(1,4)-galactosyltransferase with a sugar donor, wherein one or more
imaging agents are linked to the sugar donor, and an sugar
acceptor
thereby imaging a target cell or tissue. The polypeptide fragment
can comprise an amino acid exchange at amino acid positions 285 and
340 corresponding to human beta (1,4)-galactosyltransferase I. The
polypeptide fragment can comprise a conservative amino acid
exchange at amino acid positions 289 and 344 corresponding to
bovine beta (1,4)-galactosyltransferase I. In some examples, a
leucine (L) is exchanged for a tyrosine (Y) at amino acid position
285 and a methionine (M) is exchanged for a histidine (H) at amino
acid position 340. In other examples, a leucine (L) is exchanged
for a tyrosine (Y) at amino acid position 289 and a methionine (M)
is exchanged for a histidine (H) at amino acid position 344.
[0227] In preferred examples, the sugar donor is a UDP-galactose
analogue that comprises an azido group, a keto group, or a thiol
group that is substituted at the C2 position of galactose. The
imaging agents are linked to a sugar moiety of the sugar donor.
[0228] An imaging agent can be used according to the diagnostic or
therapeutic use as desired. For example, the imaging agent can be
selected from the group consisting of: chemical labels,
radiolabels, and fluorescent labels.
[0229] The sugar acceptor is N-acetylglucosamine (GlcNAc), and the
N-acetylglucosamine (GlcNAc) is free or attached to a peptide of a
glycopeptide.
[0230] In the imaging methods as described herein, the isolated
catalytic domain comprises SEQ ID NO: 1. The methods can take place
in the presence of magnesium.
[0231] Coupling
[0232] Methods of transfer of C2 modified galactose analogues, for
example C2 keto galactose from its UDP derivative to the GlcNAc
residue on the N-glycan chain of ovalbumin or to an
asialo-agalacto-IgG1 molecule have been described in the art, for
example in WO 2005/051429, incorporated by reference in its
entirety herein. The C2 modified galactose analogues, for example
C2 keto galactose can be biotinylated, thus allowing for
biotinylation of carriers such as ovalbumin and IgG. Methods for
transfer of by beta4 Gal-T1 enzyme have been described in the art
(Boeggeman, et al 2007).
[0233] The method of coupling a target agent to a carrier protein
via glycan chains, for example ovalbumin and IgG1, is advantageous
over other cross-linking methods. In the instant method, the target
agent is linked in a site-directed manner, only where the
carbohydrate is attached to the glycoprotein, for example as in the
IgG1 molecule at the Fe domain, away from the antigen binding site.
A problem encountered in previous approaches using monoclonal
antibodies for immunotherapy is the lack of specificity of the
reactions, resulting in heterologous labeling and a decrease in the
antibody affinity for the antigen. The instant invention overcomes
this problem.
[0234] Accordingly, the invention features methods of coupling an
agent or agents to a carrier protein. The methods as described
herein comprise incubating a reaction mixture comprising a
polypeptide fragment from a beta (1,4)-galactosyltransferase I,
wherein the polypeptide fragment comprises SEQ ID NO: 1, with a
sugar donor, and a carrier protein, in the presence of
magnesium.
[0235] The sugar donor is, in certain examples, a UDP-galactose
analogue or a UDP-GalNAc analogue that can comprise an azido group,
a keto group, or a thiol group. The azido group, the keto group or
the thiol group can be substituted at the C2 position of galactose,
thus allowing for linking of agents. Accordingly, in certain
preferred examples, one or more agents are linked to a sugar moiety
of the sugar donor. The agent can be selected from the group
consisting of: antibodies, single chain antibodies, bacterial
toxins, growth factors, therapeutic agents, targeting agents,
contrast agents, chemical labels, a radiolabels, and fluorescent
labels.
[0236] The carrier protein, in preferred examples, is ovalbumin.
The carrier protein, in other preferred examples, is an IgG. In
certain instances, it is advantageous to couple the C2
UDP-galactose analogue to biotin for detection. Subsequent
detection of biotin can be carried out by chemiluminescent assay.
The method as described herein is useful for imaging procedures,
for example in magnetic resonance imaging.
Anticoagulation
[0237] Included in the invention are applications of the
compositions of the invention as described herein are methods for
preventing platelet aggregation. Platelet aggregation refers to the
clumping together of platelets in the blood. Platelet aggregation
is part of the sequence of events leading to the formation of a
thrombus, or blood clot.
[0238] The invention describes methods for preventing platelet
aggregation comprising administering to a subject an effective
amount of a polypeptide fragment synthesized by the method
comprising incubating a reaction mixture comprising a polypeptide
fragment from a beta (1,4)-galactosyltransferase I with a sugar
donor and an sugar acceptor and thereby preventing platelet
aggregation. In the method, the polypeptide fragment may comprise
an amino acid exchange at amino acid positions 285 and 340
corresponding to human beta (1,4)-galactosyltransferase I. The
polypeptide fragment may comprise a conservative amino acid
exchange at amino acid positions 289 and 344 corresponding to
bovine beta (1,4)-galactosyltransferase I. A leucine (L) can be
exchanged for a tyrosine (Y) at amino acid position 285 and a
methionine (M) is exchanged for a histidine (H) at amino acid
position 340. A leucine (L) can be exchanged for a tyrosine (Y) at
amino acid position 289 and a methionine (M) is exchanged for a
histidine (H) at amino acid position 344.
[0239] Included in the invention are methods for preventing
platelet aggregation comprising administering to a subject an
effective amount of a polypeptide fragment synthesized by the
method comprising incubating a reaction mixture comprising a
polypeptide fragment from a beta (1,4)-galactosyltransferase 1,
wherein the polypeptide fragment comprises SEQ it) NO: 2, with a
sugar donor, wherein the sugar donor comprises a UDP-galactose
analogue and a sugar acceptor in the presence of magnesium, thereby
preventing platelet aggregation. In certain examples, the sugar
donor is a UDP-galactose analogue. In the method, the sugar
acceptor can be N-acetylglucosamine (GlcNAc). The
N-acetylglucosamine (GlcNAc) can be free or attached to a peptide
of a glycopeptide.
[0240] The method for preventing platelet aggregation can comprise
administering to a subject an effective amount of an isolated
glycoprotein synthesized by the method comprising incubating a
reaction mixture comprising an isolated catalytic domain from a
beta 1,4-galactosyltransferase I that catalyzes the formation of a
GlcNAc-beta-1,4-N-acetylgalactosamine bond and a sugar donor,
wherein the sugar donor comprises UDP-GalNAc or a UDP-GalNAc
analogue. The isolated catalytic domain can comprise SEQ ID NO:
2.
[0241] The methods for preventing platelet aggregation as described
herein are particularly useful for the treatment of diseases or
disorders where platelet aggregation is abnormal or does not occur.
For example, glanzmann thrombasthenia is a disease that is caused
by a deficiency of a protein on the surface of the platelet,
glycoprotein IIb/IIIa, and as a result, platelets do not aggregate
or clot at the site of an injury. The Bernard-Soulier syndrome is
caused by a lack or deficiency in the expression of the
glycoprotein Ib/Ix complex on the surface of the platelets. This
complex is the receptor for the von Willebrand factor (VWF). Due to
lack or deficiency of expression the binding to the VWF does not
occur at the site of vascular injury resulting in defective
platelet adhesion.
[0242] Platelets can be affected by common drugs, including asprin,
non-steroidal anti-inflammatory drugs like indomethacin, ibuprofen
and naproxen, some heart drugs, some antibiotics, blood thinners,
and antihistamines.
Therapeutic or Diagnostic Agents
[0243] A wide variety of agents may be included in the compounds of
the present invention, such as any biologically active, therapeutic
or diagnostic compound or composition. In general, the term
bioactive agent includes, but is not limited to: polypeptides,
including proteins and peptides (e.g., insulin); releasing factors
and releasing factor inhibitors, including Luteinizing Hormone
Releasing Hormone (LHRH) and gonadotropin releasing hormone (GnRH)
inhibitors; carbohydrates (e.g., heparin); nucleic acids; vaccines;
and pharmacologically active agents such as anti-infective such as
antibiotics and antiviral agents; anti-fungal agents; analgesics
and analgesic combinations; anesthetics; anorexics;
anti-helminthes; anti-arthritic agents; respiratory drugs,
including anti-asthmatic agents and drugs for preventing reactive
airway disease; anticonvulsants; antidepressants; anti-diabetic
agents; anti-diarrheals; anticonvulsants; antihistamines;
anti-inflammatory agents; toxins, anti-migraine preparations;
anti-nauseants; anticancer agents, including anti-neoplastic drugs;
anti-parkinsonism drugs; anti-pruritics; anti-psychotics;
antipyretics; antispasmodics; anticholinergics; sympathomimetics;
xanthine derivatives; cardiovascular preparations including
potassium and calcium channel blockers, beta-blockers,
alpha-blockers, cardioprotective agents; anti-arrhythmics;
anti-hyperlipidemic agents; anti-hypertensives; diuretics;
anti-diuretics; receptor agonists, antagonists, and/or mixed
function agonist/antagonists; vasodilators including general
coronary, peripheral and cerebral; central nervous system
stimulants; vasoconstrictors; cough and cold preparations,
including decongestants; enzyme inhibitors; hormones such as
estradiol, testosterone, progesterone and other steroids and
derivatives and analogs, including corticosteroids; hypnotics;
hormonolytics; immunosuppressive agents; muscle relaxants;
parasympatholytics; central nervous system stimulants; diuretics;
hypnoticsleukotriene inhibitors; mitotic inhibitors; muscle
relaxants; genetic material, including nucleic acid, RNA, DNA,
recombinant RNA, recombinant DNA, antisense RNA, antisense DNA,
hammerhead RNA, a ribozyme, a hammerheadribozyme, an antigene
nucleic acid, a ribo-oligonucleotide, a deoxyribonucleotide,
antisense ribo-oligonucleotide, and/or an antisense
deoxyribo-oligonucleotide; psychostimulants; sedatives; anabolic
agents; vitamins; herbal remedies; anti-metabolic agents;
anxiolytics; attention deficit disorder (ADD) and attention deficit
hyperactivity disorder (ADHD) drugs; neuroleptics; and
tranquilizers.
[0244] Application No. WO 2005/051429, incorporated by reference in
its entirety herein, provides a list of exemplary agents that can
be conjugated to the compositions of the instant invention.
Antibodies and Applications
[0245] As described herein, the targeting compound may be an
antibody or a fragment thereof. The term "antibody" (Ab) or
"monoclonal antibody" (Mab) is meant to include intact molecules as
well as antibody portions (e.g., Fab and F (ab') 2 portions and Fv
fragments) which are capable of specifically binding to a cell
surface marker. Such portions are typically produced by proteolytic
cleavage, using enzymes such as papain (to produce Fab portions) or
pepsin (to produce F (ab') 2 portions). Alternatively,
antigen-binding portions can be produced through the application of
recombinant DNA technology.
[0246] The immunoglobulin can be a "chimeric antibody" as that term
is recognized in the art. Also, the immunoglobulin may be a
bifunction or a hybrid antibody, that is, an antibody which may
have one arm having a specificity for one antigenic site, such as a
tumor associated antigen, while the other arm recognizes a
different target, for example, a hapten which is, or to which is
bound, an agent lethal to the antigen-bearing tumor cell.
Alternatively, the bifunctional antibody may be one in which each
arm has specificity for a different epitope of a tumor associated
antigen of the cell to be therapeutically or biologically modified,
in any case, the hybrid antibodies have a dual specificity,
preferably with one or more binding sites specific for the hapten
of choice or one or more binding sites specific for a target
antigen, for example, an antigen associated with a tumor, an
infectious organism, or other disease state.
[0247] Biological bifunctional antibodies are described, for
example, in European Patent Publication, EPA 0 105 360, which is
incorporated herein by reference. Hybrid or bifunctional antibodies
may be derived biologically, by cell fusion techniques, or
chemically, especially with cross-linking agents or disulfide
bridge-forming reagents, and may be comprised of those antibodies
and/or fragments thereof. Methods for obtaining such hybrid
antibodies are disclosed, for example, in PCT application
W083/03679, published Oct. 27, 1983, and published European
Application EPA 0 217 577, published Apr. 8, 1987, which are
incorporated herein by reference. In one embodiment, the
bifunctional antibodies are biologically prepared from a polydome
or a quadroma, or are synthetically prepared with cross-linking
agents such as bis-(maleimideo)-methyl ether ("BMME"), or with
other cross-linking agents familiar to those skilled in the
art.
[0248] In addition, the immunoglobin may be a single chain antibody
("SCA"). These may consist of single chain Fv fragments ("scFv") in
which the variable light ("V [L]") and variable heavy ("V [H]")
domains are linked by a peptide bridge or by disulfide bonds. Also,
the immunoglobulin may consist of single V [H] domains (dAbs) which
possess antigen-binding activity. See, e.g., G. Winter and C.
Milstein, Nature, 349: 295 (1991); R. Glockshuber et al.,
Biochemistry, 29: 1362 (1990); and, E. S. Ward et al., Nature, 341:
544 (1989).
[0249] The antibodies may, in certain embodiments, be chimeric
monoclonal antibodies. As used herein, the term "chimeric antibody"
refers to a monoclonal antibody comprising a variable region, i.e.,
binding region, from one source or species and at least a portion
of a constant region derived from a different source or species,
usually prepared by recombinant DNA techniques.
[0250] Chimeric antibodies comprising a murine variable region and
a human constant region are preferred in certain applications of
the invention, particularly human therapy, because such antibodies
are readily prepared and may be less immunogenic than purely murine
monoclonal antibodies. Such murine/human chimeric antibodies are
the product of expressed immunoglobulin genes comprising DNA
segments encoding murine immunoglobulin variable regions and DNA
segments encoding human immunoglobulin constant regions. Other
forms of chimeric antibodies encompassed by the invention are those
in which the class or subclass has been modified or changed from
that of the original antibody. Such "chimeric" antibodies are also
referred to as "class-switched antibodies." Methods for producing
chimeric antibodies involve conventional recombinant DNA and gene
transfection techniques well known in the art. See, e.g., Morrison,
S. L. et al., Proc. Nat'l Acad. Sci., 81: 6851 (1984).
[0251] Encompassed by the term "chimeric antibody" is the concept
of "humanized antibody," that is those antibodies in which the
framework or "complementarity" determining regions ("CDR") have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. (See,
e.g., EPA 0 239 400 (published Sep. 30, 1987)) In a preferred
embodiment, a murine CDR is grafted into the framework region of a
human antibody to prepare the "humanized antibody." See, e.g., L.
Riechmann et al., Nature, 332: 323 (1988); M. S. Neuberger et al.,
Nature, 314: 268 (1985). Furthermore, the immunoglobulin
(antibody), or fragment thereof used in the present invention may
be polyclonal or monoclonal in nature. Monoclonal antibodies are
the preferred immunoglobulins. The preparation of such polyclonal
or monoclonal antibodies is well known to those skilled in the art.
See, e.g., G. Kohler and C. Milstein, Nature, 256: 495 (1975). The
antibodies of the present invention may be prepared by any of a
variety of methods. For example, cells expressing the cell surface
marker or an antigenic portion thereof can be administered to an
animal in order to induce the production of sera containing
polyclonal antibodies. In a preferred method, a preparation of
protein is prepared and purified so as to render it substantially
free of natural contaminants. Such a preparation is then introduced
into an animal in order to produce polyclonal antisera of greater
specific activity. However, the present invention should not be
construed as limited in scope by any particular method of
production of an antibody whether bifunctional, chimeric,
bifunctional-chimeric, humanized, or an antigen-recognizing
fragment or derivative thereof.
[0252] In a preferred embodiment, the antibodies of the present
invention are monoclonal antibodies (or portions thereof). Such
monoclonal antibodies can be prepared using hybridoma technology
(Kohler et al., Nature, 256: 495 (1975); Kohler et al., Eur. J.
Immunol., 6: 511 (1976); Kohler et al, Eur. Immunol., 6: 292
(1976); Hammerling et al., In: "Monoclonal Antibodies and T-Cell
Hybridomas," Elsevier, N. Y., pp. 563-681(1981)). In general, such
procedures involve immunizing an animal (preferably a mouse) with a
protein antigen or with a protein-expressing cell (suitable cells
can be recognized by their capacity to bind antibody). The
splenocytes of such immunized mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention. After fusion,
the resulting hybridoma cells are selectively maintained in HAT
medium, and then cloned by limiting dilution as described by Wands
et al., Gastroenterology, 80: 225-232 (1981). The hybridoma cells
obtained through such a selection are then assayed to identify
clones which secrete antibodies capable of binding the antigen. In
addition, hybridomas and/or monoclonal antibodies which are
produced by such
hybridomas and which are useful in the practice of the present
invention are publicly available from sources such as the American
Type Culture Collection or commercial retailers.
[0253] The antibodies of the present invention may be labeled, for
example, for detection or diagnostic purposes, e.g., imaging.
Labels for the antibodies of the present invention include, but are
not limited to, the following: examples of enzyme labels include
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, and acetylcholine esterase; examples of radioisotopic
labels include 3H, IIIIn, I25I, I31I, 32p, 35S, 14c, 51Cr, 57To,
58Co, 59Fe, 75Se, 152Eu, 90Y, 67Cu, 217Ci, 211At, 212Pb, 47Sc, and
109Pd; examples of suitable non-radioactive isotopic labels include
I57Gd, 55Mn, 52Tr, and 56Fe; examples of fluorescent labels include
an 152 Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, aphycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label; examples of toxin labels include diphtheria toxin, ricin,
and cholera toxin; examples of chemiluminescent labels include a
luminal label, an isoluminal label, an aromatic acridinium ester
label, an imidazole label, an acridinium salt label, an oxalate
ester label, a luciferin label, a luciferase label, and an acquorin
label; and examples of nuclear magnetic resonance contrasting
agents include heavy metal nuclei such as Gd, Mn, and Fe.
Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al., Clin. Chim. Acta, 70:
1-31 (1976), and Schurs et al., Clin. Chim. Acta, 81: 1-40 (1977),
which are incorporated by reference
[0254] In one embodiment, the glycoconjugates of the invention
include monoclonal antibodies, such as those directed against tumor
antigens, for use as cancer therapeutics. Generally, monoclonal
antibodies have one N-linked bi-antennary oligosaccharide attached
at the IgG-Fe region. The terminal sugars of the oligosaccharide
moiety come in several glycoforms, for example, some are
desialated, degalactosylated, with only terminal
N-acetylglucosaminyl residues.
[0255] The monoclonal antibodies carrying only terminal
N-acetylglucosamine on the bi-antennary oligosaccharide moieties,
the Goglycoform, can be generated by de-sialylation and
de-galactosylation of the monoclonal antibodies. With the mutant
Tyr289Leu-Gal-T1(Y2891LGalT1) and UDP-a-galactose-C-2-modified, a
galactose moiety that has a chemically reactive group attached at
the C2 position of galactose, can then be transferred to Go
glycoform of the monoclonal antibody. The chemically reactive group
can include, for example, a ketone moiety that can serve as a
neutral, yet versatile chemical handle to add other agents, such as
bioactive agents, to the compound.
Methods of Treatment
[0256] The instant invention provides enzymes and methods that can
be used to promote the chemical linkage of biological molecules,
and thus provides a means to link agents for therapeutic
application. Moreover, the instant invention provides a means to
carry out the method in a physiological setting.
[0257] Accordingly, the invention features methods for the
diagnosis or treatment of a subject suffering from a disease or
disorder. The methods comprise administering to the subject an
effective amount of polypeptide fragment synthesized by the method
comprising incubating a reaction mixture comprising an isolated
catalytic domain from a beta (1,4)-galactosyltransferase I with a
sugar donor, wherein one or more agents are linked to the sugar
donor, and an sugar acceptor thereby diagnosing or treating the
subject.
[0258] The polypeptide fragment may comprise an amino acid exchange
at amino acid positions 285 and 340 corresponding to human beta
(1,4)-galactosyltransferase I. Further, a leucine (L) may be
exchanged for a tyrosine (Y) at amino acid position 285 and a
methionine (M) is exchanged for a histidine (H) at amino acid
position 340, in certain preferred embodiments.
[0259] The polypeptide fragment may comprise a conservative amino
acid exchange at amino acid positions 289 and 344 corresponding to
bovine beta (1,4)-galactosyltransferase I. Further, a leucine (L)
is exchanged for a tyrosine (Y) at amino acid position 289 and a
methionine (M) is exchanged for a histidine (H) at amino acid
position 344 in certain preferred embodiments.
[0260] In certain preferred embodiments, the polypeptide fragment
comprises SEQ ID NO: 2.
[0261] Disease states needing treatment are only limited by current
available therapeutics. As described herein, the methods of the
invention are useful for engineering of nanoparticles, including
multivalent nanoparticles, carrying any number of therapeutic
agents. For example, the nanoparticles can be used to treat cancer,
inflammatory disease, cardiovascular disease, obesity, ageing,
bacterial infection, or any other disease amenable to therapy.
[0262] The glycoconjugates compositions of the invention can be
used to treat and/or diagnose a variety of diseases and/or
disorders. For example, the glycoconjugates compositions of the
invention are used for specific, targeted delivery of bioactive
agents, including toxic drugs, agents for imaging or diagnostics,
(e.g., toxins, radionuclides), to therapeutically-relevant tissues
or cells of the body, for example, tumors. In another embodiment of
the invention, the glycoconjugates compositions of the invention
are used to deliver bioactive agents, including DNA vectors, to
cells.
[0263] As further examples, the glycoconjugates compositions of the
invention are useful for the treatment of a number of diseases
and/or disorders including, but not limited to: cancer, both solid
tumors as well as blood-borne cancers, such as leukemia;
hyperproliferative disorders that can be treated by the compounds
of the invention include, but are not limited to, neoplasms located
in the: abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
(central and peripheral), lymphatic system, pelvic, skin, soft
tissue, spleen, thoracic, and urogenital.
[0264] The glycoconjugates of the invention can be used to treat
cardiovascular diseases and disorders including, but not limited
to, myocardial infarction (heart attack), cerebrovascular diseases
(stroke), transient ischaemic attacks (TIA), peripheral vascular
diseases, arteriosclerosis, angina, high blood pressure, high
cholesterol, arrhythmia.
[0265] The glycoconjugates of the invention can be used to treat
genetic diseases, such as enzyme deficiency diseases.
[0266] The glycoconjugates of the invention can be used to treat
hyperproliferative disorders. Examples of such hyperproliferative
disorders that can be treated by the glycoconjugates of the
invention are as described in Application WO 2005/051429, and are
incorporated by reference in its entirety herein.
[0267] The glycoconjugates of the present invention are also useful
for raising an immune response against infectious agents. Viruses
are one example of an infectious agent that can cause disease or
symptoms that can be treated by the compounds of the invention.
Examples of viruses that can cause disease or symptoms and that can
be treated by the glycoconjugates of the invention are as described
in Application WO 2005/051429, and are incorporated by reference in
its entirety herein.
[0268] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated by the glycoconjugates of the
invention are as described in Application WO 2005/051429, and are
incorporated by reference in its entirety herein.
[0269] Additionally, the glycoconjugates of the invention are
useful for treating autoimmune diseases. An autoimmune disease is
characterized by the attack by the immune system on the tissues of
the victim. Autoimmune disease is characterized by the inability of
the recognition of "self" and the tissue of the afflicted subject
is treated as a foreign target. The compounds of the present
invention are therefore useful for treating autoimmune diseases by
desensitizing the immune system to these self antigens by provided
a TCR signal to T cells without a costimulatory signal or with an
inhibitory signal. Examples of autoimmune diseases which may be
treated using the glycoconjugates of the present invention are as
described in Application WO 2005/051429, and are incorporated by
reference in its entirety herein.
[0270] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by glycoconjugates of the invention. Moreover, the
glycoconjugates of the invention can be used to treat anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[0271] The glycoconjugates of the invention which can inhibit an
immune response are also useful for treating and/or preventing
organ rejection or graft versus host disease, atherosclerosis;
olitis; regional enteritis; adult respiratory distress syndrome;
local manifestations of drug reactions, such as dermatitis, etc.;
inflammation-associated or allergic reaction patterns of the skin;
atopic dermatitis and infantile eczema; contact dermatitis;
psoriasis; lichen planus; allergic enteropathies; allergic
rhinitis; bronchial asthma; hypersensitivity or destructive
responses to infectious agents; poststreptococcal diseases, e.g.,
cardiac manifestations of rheumatic fever, and the like.
Vaccines
[0272] The invention also provides methods for eliciting an immune
response in a mammal such as a human, including administering to a
subject an immunological composition comprising a compound or
composition as described herein. Therefore, one embodiment of the
present invention is to use the glycoconjugates described herein in
an immunological preparation.
[0273] The immunological composition according to the instant
invention may be prepared by any method known in the art. For
example, glycoconjugates of the present invention are prepared and
are then injected into an appropriate animal. The compositions
according to the present invention may be administered in a single
dose or they may be administered in multiple doses, spaced over a
suitable time scale to fully utilize the secondary immunization
response. For example, antibody titers may be maintained by
administering boosters once a month. The vaccine may further
comprise a pharmaceutically acceptable adjuvant, including, but not
limited to Freund's complete adjuvant, Freund's incomplete
adjuvant, lipopolysaccharide, monophosphoryl A, muramyl dipeptide,
liposomes containing lipid A, alum, muramyl
tripeptide-phosphatidylethanoloamine, keyhole and limpet
hemocyanin.
Administration
[0274] The compositions of the present invention may be
administered by any means that results in the contact of the
bioactive agent with the agent's site or site(s) of action on or in
a subject, e. g., a patient. The compositions may be administered
alone or in conjunction with one or more other therapies or
treatments.
[0275] The targeted glycoconjugates produced according to the
present invention, can be administered to a mammalian host by any
route. Thus, as appropriate, administration can be orally,
intravenously, rectally, parenterally, intracistemally,
intradermally, intravaginally, intraperitoneally, topically (as by
powders, ointments, wets, creams, drops or transdermal patch),
bucally, or as an oral or nasal spray. The term "parenteral" as
used herein refers to modes of administration which include
intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion. Parenteral
administration in this respect includes administration by the
following routes: intravenous, intramuscular, subcutaneous,
intraocular, intrasynovial, transepithelial including transdermal,
ophthalmic, sublingual and buccal; topically including ophthalmic,
dermal, ocular, rectal and nasal inhalation via insufflation,
aerosol and rectal systemic.
[0276] In addition, administration can be by periodic injections of
a bolus of the therapeutic or can be made more continuous by
intravenous or intraperitoneal administration from an external
source. In certain embodiments, the therapeutics of the instant
invention can be pharmaceutical-grade and in compliance with the
standards of purity and quality control required for administration
to humans. Veterinary applications are also within the intended
meaning as used herein.
[0277] The formulations, both for veterinary and for human medical
use, of the therapeutics according to the present invention
typically include such therapeutics in association with a
pharmaceutically acceptable carrier therefor and optionally other
ingredient (s). The carrier (s) can be acceptable in the sense of
being compatible with the other ingredients of the formulations and
not deleterious to the recipient thereof. Pharmaceutically
acceptable carriers are intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration.
[0278] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such asethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. pH can
be adjusted with acids or bases, such as hydrochloric acid or
sodium hydroxide.
[0279] Useful solutions for oral or parenteral administration can
be prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences. Formulations for parenteral administration also can
include glycocholate for buccal administration, methoxysalicylate
for rectal administration, or citric acid for vaginal
administration. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0280] Formulations of the present invention suitable for oral
administration can be in the form of discrete units such as
capsules, gelatin capsules, sachets, tablets, troches, or lozenges,
each containing a predetermined amount of the drug; in the form of
a powder or granules; in the form of a solution or a suspension in
an aqueous liquid or non-aqueous liquid; or in the form of an
oil-in-water emulsion or a water-in-oil emulsion. The therapeutic
can also be administered in the form of a bolus, electuary or
paste. A tablet can be made by compressing or molding the drug
optionally with one or more accessory ingredients. Compressed
tablets can be prepared by compressing, in a suitable machine, the
drug in a free-flowing form such as a powder or granules,
optionally mixed by a hinder, lubricant, inert diluent, surface
active or dispersing agent. Molded tablets can be made by molding,
in a suitable machine, a mixture of the powdered drug and suitable
carrier moistened with an inert liquid diluent.
[0281] Oral compositions generally include an inert diluent or an
edible carrier.
[0282] For the purpose of oral therapeutic administration, the
active compound can be incorporated with excipients. Oral
compositions prepared using a fluid carrier for use as a mouthwash
include the compound in the fluid carrier and are applied orally
and swished and expectorated or swallowed. Pharmaceutically
compatible binding agents, and/or adjuvant materials can be
included as part of the composition. The tablets, pills, capsules,
troches and the like can contain any of the following ingredients,
or compounds of a similar nature; a binder such as microcrystalline
cellulose, gumtragacanth or gelatin; an excipient such as starch or
lactose; a disintegrating agent such as alginic acid, Primogel, or
corn starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0283] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition can
be sterile and can be fluid to the extent that easy syringability
exists. It can be stable under the conditions of manufacture and
storage and can be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, and sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent which delays
absorption, for example, aluminum monostearate and gelatin.
[0284] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization, e.g.,
filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
methods of preparation include vacuum drying and freeze-drying
which yields a powder of the active ingredient plus any additional
desired ingredient.
[0285] Formulations suitable for topical administration, including
eye treatment, include liquid or semi-liquid preparations such as
liniments, lotions, gels, applicants, oil-in-water or water-in-oil
emulsions such as creams, ointments or pasts; or solutions or
suspensions such as drops. Formulations for topical administration
to the skin surface can be prepared by dispersing the therapeutic
with a dermatologically acceptable carrier such as a lotion, cream,
ointment or soap. In some embodiments, useful are carriers capable
of forming a film or layer over the skin to localize application
and inhibit removal.
[0286] For inhalation treatments, such as for asthma, inhalation of
powder (self-propelling or spray formulations) dispensed with a
spray can, a nebulizer, or an atomizer can be used. Such
formulations can be in the form of a finely comminuted powder for
pulmonary administration from a powder inhalation device or
self-propelling powder-dispensing formulations. In the case of
self-propelling solution and spray formulations, the effect can be
achieved either by choice of a valve having the desired spray
characteristics (i.e., being capable of producing a spray having
the desired particle size) or by incorporating the active
ingredient as a suspended powder in controlled particle size. For
administration by inhalation, the therapeutics also can be
delivered in the form of an aerosol spray from a pressured
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulize. Nasal drops also can
be used.
[0287] Systemic administration also can be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants generally are known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and filsidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the therapeutics
typically are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0288] The therapeutics can be prepared with carriers that will
protect against rapid elimination from the body, such as a
controlled release formulation, including implants and
microencapsulated delivery systems.
[0289] The compounds of the invention may also suitably be
administered by sustained-release systems. Suitable examples of
sustained-release compositions include Semi-permeable polymer
matrices in the form of shaped articles, e.g., films, or
microcapsules. Sustained-release matrices include polylactides
(U.S. Pat. No. 3,773,919, EP 58, 481), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:
547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et
al., J. Biomed. Mater. Res. 15: 167-277 (1981), and R. Langer,
Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate(R. Langer et
al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP) 133,988).
Sustained-release compositions also include liposomally entrapped
compositions of the present invention (Epstein, et al., Proc. Natl.
Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl.
Acad. Sci, USA 77: 4030-4034 (1980).
[0290] The compositions can be formulated in dosage unit form for
ease of administration and uniformity of dosage. Dosage unit form
refers to physically discrete units suited as unitary dosages for
the subject to be treated; each unit containing a predetermined
quantity of active compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0291] Generally, the therapeutics identified according to the
invention can be formulated for administration to humans or other
mammals, for example, in therapeutically effective amounts, e.g.,
amounts which provide appropriate concentrations of the bioactive
agent to target tissue/cells for a time sufficient to induce the
desired effect. Additionally, the therapeutics of the present
invention can be administered alone or in combination with other
molecules known to have a beneficial effect on the particular
disease or indication of interest. By way of example only, useful
cofactors include symptom-alleviating cofactors, including
antiseptics, antibiotics, antiviral and antifungal agents and
analgesics and anesthetics.
[0292] The effective concentration of the therapeutics identified
according to the invention that is to be delivered in a therapeutic
composition will vary depending upon a number of factors, including
the final desired dosage of the drug to be administered and the
route of administration. The preferred dosage to be administered
also is likely to depend on such variables as the type and degree
of the response to be achieved; the specific composition of another
agent, if any, employed; the age, body weight, general health, sex
and diet of the patient; the time of administration, route of
administration, and rate of excretion of the composition; the
duration of the treatment; bioactive agent (such as a
chemotherapeutic agent) used in combination or coincidental with
the specific composition; and like factors well known in the
medical arts. In some embodiments, the therapeutics of this
invention can be provided to an individual using typical dose units
deduced from the earlier-described mammalian studies using
non-human primates and rodents. As described above, a dosage unit
refers to a unitary, i.e. a single dose which is capable of being
administered to a patient, and which can be readily handled and
packed, remaining as a physically and biologically stable unit dose
comprising either the therapeutic as such or a mixture of it with
solid or liquid pharmaceutical diluents or carriers.
[0293] Therapeutics of the invention also include "prodrug"
derivatives. The term prodrug refers to a pharmacologically
inactive (or partially inactive) derivative of a parent molecule
that requires biotransformation, either spontaneous or enzymatic,
within the organism to release or activate the active component.
Prodrugs are variations or derivatives of the therapeutics of the
invention which have groups cleavable under metabolic conditions.
Prodrugs become the therapeutics of the invention which are
pharmaceutically active in vivo, when they undergo solvolysis under
physiological conditions or undergo enzymatic degradation. Prodrug
forms often offer advantages of solubility, tissue compatibility,
or delayed release in the mammalian organism (see, Bundgard, Design
of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985 and
Silverman, The Organic Chemistry of Drug Design and Drug Action,
pp. 352-401, Academic Press, San Diego, Calif., 1992),
Kits
[0294] Also included in the invention are kits. Preferably, kits
comprise a packaging material, and a polypeptide fragment from a
beta (1,4)-galactosyltransferase I according to any one of the
aspects of the invention as described herein. The kits, in certain
preferred embodiments, comprise a sugar donor. The donor can be any
one of UDP-galactose, UDP-GalNAc, UDP-GalNAc analogues or
UDP-Galactose analogues. The kits can also comprise an agent. In
preferred examples, the agent is linked to the sugar donor.
Exemplary agents are described in this disclosure. Certain agents
can be selected from antibodies, single chain antibodies, bacterial
toxins, growth factors, therapeutic agents, contrast agents,
targeting agents, chemical labels, a radiolabels, and fluorescent
labels.
EXAMPLES
[0295] It should be appreciated that the invention should not be
construed to be limited to the examples that are now described;
rather, the invention should be construed to include any and all
applications provided herein and all equivalent variations within
the skill of the ordinary artisan.
[0296] As described in more detail below, the experiments reported
herein are based on the finding that the genetically engineered
beta Gal-TI enzyme, Gal-TI-Y289L-M344H, which can transfer GalNAc
or 2' modified galactose to GlcNAc residue of a glycan chain in the
presence of magnesium instead of manganese, is very useful for the
synthesis of a disaccharide unit with a chemical handle on the
glycan chain of glycoconjugates on live cells. Since, in contrast
to manganese, the concentration of magnesium used is not toxic to
cells, the galactose with a chemical handle transferred by the
mutant enzyme can be used for detection of available GlcNAc
residues on a given live cell under different physiological states
or for conjugation of pharmacological agents for therapeutic
purposes.
Example 1
The GalT1-Y289L-M34411 Double Mutant Transfers Sugar in the
Presence of Mg2+
[0297] X-ray crystal structures of the catalytic domain of many
glycosyltransferases have been determined in recent years, and
these studies show that the specificity of the sugar donor is
determined by residues in the sugar-nucleotide binding pocket of
glycosyltransferases. This structural information has made it
possible to reengineer the existing glycosyltransferases. For
example, the beta 1,4-galactosyltransferase family in vertebrates
(beta 4Gal-T1 to T7) is responsible for the transfer of galactose
from the donor UDP-galactose (UDP-Gal) to various glycans in a beta
1-4 linkage (Amado et al, 1999; Hennet et al., 2002)). The residue
Tyr289 (or Phe) in the catalytic pocket of b4Gal-T1, determines the
sugar donor substrate specificity of the enzyme toward UDP-Gal
(Qasba et al., 2005; Ramakrishnan et al. 2002). Mutation of Tyr289
to Leu or Ile enlarges the binding pocket such that the mutant
enzyme, beta4Gal-T1-Y289L, has beta
1,4-Nacetylgalactosaminyltransferase (beta 4GalNAc-T) activity,
which is as efficient as its beta1,4-galactosyltransferase (beta
4Gal-T) activity (Ramakrishnan et al. 2002; Ramakrishnan et al,
2004). Similarly, in blood group A GalNAc-transferase, the residues
Leu266 and Gly268, and in blood group B Gal transferase, the
residues Met266 and Ala268 determine the specificities toward their
respective sugar donors, UDP-GalNAc and UDP-Gal (Marcus et al.
2003). Mutation Met266 to Leu266 in blood group B Gal-transferase
changes the enzyme sugar donor specificity toward UDP-GalNAc.
Furthermore, in beta 1,3-glucuronyltransferases-1 (GlcA-T1), His
308 (His 311 in GlcAT-P) (Pederson et al, 2002; Kakuda et al.,
2004) determines the enzyme specificity toward the sugar donor
UDP-GlcUA. Mutation of His308 to Arg308 changes the specificity of
the sugar donor to UDP-Glc, UDP-Man, or UDP-GlcNAc.
[0298] It has previously been shown that the sugar donor
specificity of beta 4Gal-T1 toward UDP-Gal is determined by a
single amino acid, Tyr, at position 289 (Ramakrishnan, B., et al.
2002). When Tyr289 is mutated to Leu, the sugar donor specificity
of aGal-T1 is broadened in a way that, in contrast to the wild-type
enzyme which lacks GalNAc-T activity, the mutant beta
4Gal-T1-Y2891, exhibits both beta 4Gal-T and beta 4GalNAc-T
activities. The Tyr289 mutant can also transfer from the UDP
derivatives the galactose moiety that has, at the C2 position,
substitutions other than the 2-N-acetyl group (--NH--CO--CH3 in
GalNAc) (21). Described herein is the transfer of GalNAc to
N-glycans glycoproteins by a GalT1-Y289L-M344-H double mutant.
[0299] The instant invention describes a novel GalT1-Y289L-M3448
double mutant that is still functional, that is the
GalT1-Y289L-M344H double mutant is able to transfer sugar in the
presence of magnesium.
[0300] Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence
of manganese ion (Mn2+) transfers galactose from UDP-galactose
(UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or
linked to an oligosaccharide. Crystallographic studies on bovine
beta4Gal-T1 have shown that the primary metal binding site is
located in the hinge region of a long flexible loop, which upon Mn
(2+) and UDP-Gal binding changes from an open to a closed
conformation. This conformational change creates an oligosaccharide
binding site in the enzyme.
[0301] The results presented here demonstrate transfer of GalNAc or
a modified sugar by the double mutant, beta 4Gal-T1_M340H_Y285L, in
the presence of Mg2+. This is in contrast to the single mutant
b4Gal-T1Y289L or the wild type b4Gal-T1, which both require the
presence of Mn2+ for the transfer of GalNAc or a modified sugar.
The experiments shown in FIG. 1 were performed with the double
mutant, b4Gal-T1_M340H_Y285L, which was constructed from the single
mutants, b4Gal-T1_M340H and b4Gal-T1_Y285L. The b4Gal-T1_M340H
single mutant requires Mg2+ for the transfer reaction, while the
b4Gal-T1_Y285L single mutant requires Mn2+ for the transfer
reaction. In FIGS. 1, (A), (B) and (C) show the MALDI mass spectra
of glycans after the transfer of GalNAc (shown in B)
2-keto-galactose (shown in C) to the sugar acceptor,
heptasaccharide tetrapeptide (A), Arg
[GlcNAc.beta.1,2-Man.alpha.1,6-(GlcNAc.beta.1,2-Man.alpha.1,3)-Man.beta.1-
,4-GlcNAc.beta.1,4-GlcNAc.beta.]-Asn-Glu-Gly, by the double mutant
enzyme, b4Gal-T1M340H_Y285L. In FIG. 1, major peaks are annotated
with the carbohydrate structure shown in the symbols for
monosaccharides, according to the nomenclature adopted by the
consortium for functional glycomics (publicly available on the
world wide web at functional glycomics.org/static/consortium/).
Panel (A) shows a peak at 1773.9 m/z corresponding to the starting
branched heptasaccharide tetrapeptide. Panel (B) shows a peak at
2179.0 tri/z corresponding to a nanosaccharide tetrapeptide having
two added GaINAc moieties as indicated and Panel (C) the peak at
2178 m/z corresponding to a nanoasaccharide tetrapeptide having two
added 2-keto-galactose moieties as indicated.
[0302] FIG. 2 shows the specific activities of the catalytic domain
of the human b4Gal-T1-M340H-Y285L in the presence of manganese or
magnesium. The reactions were performed under saturating conditions
of all substrates. The results presented in FIG. 2 show that the
catalytic, domain of the human b4Gal-T1-M340H-Y285L is active in
the presence of magnesium. This is in contrast to the wild type,
which is not active (not able to transfer sugar) in the presence of
magnesium.
Example 2
GalT1-Y289L-M344H Prevents Platelet Aggregation
[0303] The double mutant enzyme GalT1-Y289L-M344H has use as an
anticoagulation agent. It was found that the double mutant enzyme
can transfer galactose using 50-75 micromolar concentrations of
UDP-galactose in the presence of magnesium to platelets and thus
prevent their aggregation in the cold. In contrast, the wild type
enzyme requires 800 micromolar concentrations of UDP-galactose at
high concentrations of Ca2+ with a low concentration of manganese
(data not shown).
Methods
[0304] The invention was performed using the following methods:
Met344His Mutant
[0305] Site-directed mutagenesis was performed using the PCR
method. Construction of the mutants was carried out as described
previously in Qasba et al. (Biochemistry 2004, 43, 12513-12522),
incorporated by reference in its entirety herein.
Bacterial Growth and Plasmid Transformation
[0306] Bacterial growth and plasmid transformations can be
performed using standard procedures (Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley-Interscience, New York (1987)). US Published Application
20060084162, incorporated by reference in its entirety herein,
describes methods for bacterial growth and transformation using the
plasmid pEGT-d129, which encodes the catalytic domain (residues
130-402) of bovine.beta.(1,4)-galactosyltransferase I.
Site-directed mutagenesis can be performed using a CLONTECH
site-directed mutagenesis transformer kit. Thus, the transformation
mixture contains the template pEGT-d129, a selection primer, and a
mutagenic primer for creation of a desired mutant. Mutants are
screened for the incorporated mutations by looking for changes in
restriction enzyme digestion patterns and confirmed by DNA
sequencing. The positive clones were transformed into
B834(DE3)pLysS cells.
Expression and Purification of Inclusion Bodies
[0307] The expression and purification of the inclusion bodies can
be carried out as described previously (Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley-Interscience, New York (1987)). The inclusion bodies are
S-sulfonated by dissolving in 5 M GdnHCl, 0.3 M sodium sulfite, and
the addition of di-sodium 2-nitro-5-thiosulfobenzoate to a final
concentration of 5 mM. The sulfonated protein is precipitated by
dilution with water, and the precipitate was washed thoroughly.
[0308] Briefly, 100 mg of sulfonated protein is folded in one liter
folding solution for 48 hours. Inclusion of 10% glycerol and 10 mM
lactose in the folding solution enhances the folding efficiency of
the galactosyltransferase, e.g. beta-1,4-galactosyltransferase
(beta4Gal-T1). After refolding the protein, the folding solution is
extensively dialyzed against water. During dialysis the misfolded
protein precipitates out, while the folded protein remains soluble.
The soluble protein is first concentrated and then purified a
Ni-column. Nearly 2 mg of folded ppGalNAc-T2 protein is obtained
form 1 liter of folding solution. Purified protein may be tested
for catalytic activity using a 13 amino acid peptide,
PTTDSTTPAPTTK, as an acceptor using methods described previously
(Fritz, T. A et al. J. Biol Chem. 2006).
[0309] Improving the folding conditions: In recent years factorial
folding screens (Rudolph and Lilie, FASEB J., 10:40-56 (1996); Chen
and Gouaux, Proc. Natl. Acad. Sci., 94:13431-13436 (1997);
Armstrong et al., Prot. Sci., 8:1475-1483 (1999)) have been
developed for examining the folding efficiencies of proteins from
inclusion bodies. To improve the in vitro folding efficiency, 8
different folding conditions similar to the formulations described
in the Foldlt Screen kit (Hampton Research, Calif.) with certain
modifications were tested. Condition I: 50 mM Tris-HCl pH 8.0, 5 mM
EDTA, 0.5 M guanidine-HCl, 8 mM cysteamine and 4 mM cystamine.
Condition II: 55 Mes pH 6.5, 10.56 mM NaCl, 0.44 mM KCl, 2.2 mM
MgCl.sub.2, 2.2 mM CaCl.sub.2, 0.5 guanidine-HCl. Condition III:
similar to condition II with respect to the buffer, pH, chaotrope
and salt condition, but it had 0.055% PEG-4000, 1.1 mM EDTA, 0.44 M
sucrose and 0.55 M L-arginine. Condition IV: 55 mM Mes pH 6.5, 264
mM NaCl, 11 mM KCl, 0.055% PEG-4000, 0.5 M guanidine-HCl, 2.2 mM
MgCl2, 2.2 mM CaCl.sub.2 and 0.44 M sucrose. Condition V: 55 mM
Tris pH 8.2, 10.56 mM NaCl, 0.44 mM KCl, 1.1 mM EDTA, 0.44 M
sucrose. Conditions VI and VIII are similar except for the presence
of redox agents. Condition VII: 55 mM Mes pH 6.5, 264 mM NaCl, 11
mM KCl, 1.1 mM EDTA, 0.5 M guanidine-HCl, and 0.55 M L-arginine.
The buffers II through VII had 100 mM GSH and 10 mM GSSG.
Conditions I and VIII, had 8 mM cysteamine and 4 mM cystamine.
Condition VIII, gave the highest enzymatic activity, soluble and
folded protein, was 50 mM Tris-HCl pH 8.0, 10.56 mM NaCl, 0.44 mM
KCl, 2.2 mM MgCl2, 2.2 mM CaCl.sub.2 0.5 M guanidine-HCl, 8 mM
cysteamine and 4 mM cystamine, 0.055% PEG-4000 and 0.55
ML-arginine.
Mutation
[0310] Mutation of certain amino acid residues as described herein
is, in certain examples, performed site-directed mutagenesis. US
Published Application 20060084162 describes methods for site
directed mutagensis of amino acid position 289 of the
bovine.beta.(1,4)-galactosyltransferase I, performed using the PCR
method.
Gal-T and GalNAc-T Enzyme Assays
[0311] Gal-T and GalNAc T enzyme assays are easily performed
according to methods described in the art, for example US Published
Application 20060084162. Protein concentrations are measured using
the Bio-Rad protein assay kit, based on the method of Bradford and
further verified on SDS gel. An in vitro assay procedure for the
Gal-T1 has been reported previously (Ramakrishnan et al., J. Biol.
Chem., 270, 87665-376717 (2000). The activities were measured using
UDP-Gal or UDP-GalNAc as sugar nucleotide donors, and GlcNAc and
Glc as the acceptor sugars. For the specific activity measurements,
a 100-.mu.l incubation mixture containing 50 mM
.beta.-benzyl-GlcNAc, 10 mM MnCl.sub.2, 10 mM Tris-HCl, pH 8.0, 500
.mu.M UDP-Gal or UDP-GalNAc, 20 ng of Gal-T1, and 0.5 .mu.Cl of
[.sup.3H]UDP-Gal or [.sup.3H]UDP-GalNAc was used for each Gal-T or
GalNAc-T reaction. The incubation was carried out at 37.degree. C.
for 10 min. The reaction was terminated by adding 200 .mu.l of cold
50 mM EDTA, and the mixture was passed through a 0.5-ml bed volume
column of AG1-X8 cation resin (Bio-Rad) to remove any unreacted
[.sup.3H]UDP-Gal or [.sup.3H]UDP-GalNAc. The column was washed
successfully with 300, 400, and 500 .mu.l of water, and the column
flow-through was diluted with Biosafe scintillation fluid;
radioactivity was measured with a Beckman counter. A reaction
without the acceptor sugar was used as a control. A similar assay
was carried out to measure the GalNAc-T activity with Glc and other
acceptors in the presence of 50 .mu.M bovine LA (Sigma).
[0312] The in vitro assay for enzyme activity (beta Gal T1, double
mutant beta-gal) can be performed as described (Boeggeman et al.,
Glycobiology, 12:395-407 (2002)). The .sup.3H-labeled-UDP-Gal or
UDP-Galactose was used as sugar donor and GlcNAc as the sugar
acceptor. A reaction without GlcNAc was used as a control.
Other Embodiments
[0313] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0314] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0315] All patents and publications mentioned in this specification
are herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and
individually indicated to be incorporated by reference.
REFERENCES
[0316] Amado, M., Almeida, R., Schwientek, T., and Clausen, H.
(1999) Identification and characterization of large
galactosyltransferase gene families: galactosyltransferases for all
functions. Biochim. Biophys. Acta 1473, 35-53. [0317] Bell, J. E.,
Beyer, T. A., and Hill, R. (1976) The kinetic mechanism of bovine
milk galactosyltransferase. The role of R-laetalbumin, J. Biol.
Chem. 251, 3003-3013. [0318] Berger, E. G. and Rohrer, J. (2003)
Galactosyltransferase--still up and running. Biochimie 85, 261-274.
[0319] Boeggeman, E., and Qasba, P. K. (2002) Studies on the metal
binding sites in the catalytic domain of a
1,4-galactosyltransferase, Glycobiology 12, 395-407. [0320]
Boeggeman, E., Balaji, P. V., Sethi, N., Masibay, A. S., and Qasba,
P. K. (1993) Expression of deletion constructs of bovine
beta-1,4-galactosyltransferase in Escherichia coli: importance of
Cys134 for its activity, Protein Eng. 6, 779-785. [0321] Boeggeman,
E. E., Ramakrishnan, B. and Qasba, P. K. The N-terminal stem region
of bovine and human beta1,4-galactosyltransferase 1 increases the
in vitro folding efficiency of their catalytic domain from
inclusion bodies. Protein Expr Purif. (2003) 30, 219-29. [0322]
Boeggeman E. E., Ramakrishnan B., Kilgore C., Khidekel N.,
Hsieh-Wilson L. C., Simpson J. T., and Qasba P. K. Direct
Identification of Nonreducing GlcNAc Residues on N-Glycans of
Glycoproteins Using a Novel Chemoenzymatic Method. Bioconjugate
Chem. 2007, 18, 806-814. [0323] Boix, E. et al. (2001) Structure of
UDP complex of UDP-galactose:
b-galactoside-a-1,3-galactosyltransferase at 1.53-A.degree.
resolution reveals a conformational change in the catalytically
important C terminus. J. Biol. Chem. 276, 48608-48614 [0324] Brew,
K., Vanaman, T. C., and Hill, R. L. (1968) The role of
R-lactalbumin and the A protein in lactose synthetase: a unique
mechanism thr the control of a biological reaction, Proc. Natl.
Acad. Sci. U.S.A. 59, 491-497. [0325] Brodbeck, U., Denton, W. L.,
Tanahashi, N., and Ebner, K. E. (1967) The isolation and
identification of the B protein of lactose synthetase
R-lactalbumin, J. Biol. Chem. 242, 1391-1397. [0326] Fritz. T. A.,
Raman, J., and Tabak, L. A. Dynamic association between the
catalytic and lectin domains of human UDP-GalNAc:polypeptide
alpha-N-acetylgalactosaminyltransferases-2. J Biol Chem. (2006)
281, 8613-9. [0327] Gastinel, L. N., Cambillau, C., and Bourne, Y.
(1999) Crystal structures of the bovine a4-galactosyltransferase
catalytic domain and its complex with uridine diphosphogalactose,
EMBO J. 18, 3546-3557. [0328] Geren, C. R., Magee, S. C., and
Ebner, K. E. (1975) Circular dichroism changes in
galactosyltransferase upon substrate binding, Biochemistry 14,
1461-1463. [0329] Gunasekaran, K., Buyong, M., Ramakrishnan, B.,
Qasba, P. K., and Nussinov, R. (2003) Interdependence of backbone
flexibility, residue conservation, and enzyme function: a case
study on beta1,4-galactosyltransferase-I, Biochemistry 42,
3674-3687. [0330] Hennet, T. (2002) The galactosyltransferase
family. Cell. Mol. Life Sci. 59, 1081-1095. [0331] Hu, Y. et al.
(2003) Crystal structure of the MurG:UDP-GlcNAc complex reveals
common structural principles of a superfamily of
glycosyltransferases. Proc. Natl. Acad. Sci. U.S.A. 100, 845-849
[0332] Kakuda, S., Shiba, T., Ishiguro, M., Tagawa, H., Oka, S.,
Kajihara, Y., Kawasaki, T., Wakatsuki, S., and Kato, R. (2004)
Structural basis for acceptor substrate recognition of a human
glucuronyltransferase, GlcAT-P, an enzyme critical in the
biosynthesis of the carbohydrate epitope HNK-1. J. Biol. Chem. 279,
2.2.693-22703. [0333] Lobsanov, Y. D. et al. (2004) Structure of
Kre2p/Mnt1p: a yeast a 1,2-mannosyltransferase involved in
mannoprotein biosynthesis. J. Biol. Chem. 279, 17921-17931 [0334]
Lowe, J. B., and Marth, J. D. (2003) A genetic approach to
mammalian glycan function. Annu. ReV. Biochem. 72, 643-691. [0335]
Marcus, S. L., Polakowski, R., Seto, N. O. L., Leinala, E.,
Borisova, S., Blancher, A., Roubinet, F., Evans, S. V., and Palcic,
M. M. (2003) A single point mutation reverses the donor specificity
of human blood group B-synthesizing galactosyltransferase. J. Biol.
Chem. 278, 12403-12405. [0336] Morera, S. et al. (1999) T4 phage
b-glucosyltransferase: substrate binding and proposed catalytic
mechanism. J. Mol. Biol. 292, 717-730. [0337] Mulichak, A. M. et
al. (2001) Structure of the UDP-glucosyltransferase GtfB that
modifies the heptapeptide aglycone in the biosynthesis of
vancomycin group antibiotics. Structure 9, 547-557. [0338] Negishi,
M. et al, (2003) Glucosaminylglycan biosynthesis; what we can learn
from the X-ray crystal structures of glycosyltransferases GlcAT1
and EXTL2. Biochem. Biophys. Res. Commun. 303, 393-398. [0339]
Powell, J. T., and Brew, K. (1976) Metal ion activation of
galactosyltransferase, J. Biol. Chem. 251, 3645-3652. [0340]
Pedersen, L. C., Darden, T. A., and Negishi, M. (2002) Crystal
structure of beta 1,3glucuronyltransferase I in complex with active
donor substrate UDP-GlcUA. J. Biol. Chem. 277, 21869-21873. [0341]
Powell, J. T., and Brew, K. (1976) A comparison of the interactions
of galactosyltransferase with a glycoprotein substrate (ovalbumin)
and with R-lactalbumin, J. Biol. Chem. 251, 3653-3663, [0342]
Qasba, P. K., Ramakrishnan, B., and Boeggeman, E. (2005)
Substrate-induced conformational changes in glycosyltransferases.
Trends Biochem. Sci. 30, 53-62. [0343] Ramakrishnan, B.,
|Boeggeman, E, and Qasba P. K. (2004), Effect of the Met344His
Mutation on the Conformational Dynamics of Bovine
beta-1,4-Galactosyltransferase: Crystal Structure of the Met344His
Mutant in Complex with Chitobiose. Biochemistry 2004, 43,
12513-12522. [0344] Ramakrishnan, B., and Qasba, P. K. (2001)
Crystal structure of lactose synthase reveals a large
conformational change in its catalytic component, the
beta1,4-galactosyltransferase-1, J. Mol. Biol. 310, 205-218. [0345]
Ramakrishnan, B., Shah, P. S., and Qasba, P. K. (2001a)
R-Lactalbumin (LA) stimulates milk beta-1,4-galactosyltransferase I
(beta4Gal-T1) to transfer glucose from UDP-glucose to
N-acetylglucosamine. Crystal structure of beta4Gal-T1aLA complex
with UDP-Glc, J. Biol. Chem. 276, 37665-37671. [0346] Ramakrishnan,
B., and Qasba, P. K. (2002) Structure-based design of
beta1,4-galactosyltransferase I (beta4Gal-T1) with equally
efficient N-acetylgalactosaminyltransferase activity: point
mutation broadens beta4Gal-T1 donor specificity, J. Biol. Chem.
277, 20833-20839. [0347] Ramakrishnan, B., Balaji, P. V., and
Qasba, P. K. (2002a) Crystal structure of beta
1,4-galactosyltransferase complex with UDP-Gal reveals an
oligosaccharide acceptor binding site, J. Mal. Biol. 318, 491-502,
[0348] Ramakrishnan, B., and Qasba, P, K. (2003) Comparison of the
closed conformation of the beta,1,4-galactosyltransferase-1
(beta4Gal-T1) in the presence and absence of R-lactalbumin (LA), J.
Biomol. Struct. Dyn. 21, 1-8. [0349] Ramakrishnan, B., Boeggeman,
E., Ramasamy, V., and Qasba, P. K. (2004a) Structure and catalytic
cycle of beta-1,4-galactosyltransferrase. Curr. Opin. Struct. Biol.
14, 593-600 [0350] Raman, R., Sasisekharan, V., and Sasisekharan,
R. (2005) Structural insights into biological roles of
protein-glycosaminoglycan interactions. Chem. Biol. 12, 267-277.
[0351] Ramasamy, V., Ramakrishnan, B., Boeggeman, E., and Qasba, P.
K. (2003) The role of tryptophan 314 in the conformational changes
of a1,4-galactosyltransferase-I, J. Mol. Biol. 331, 1065-1076.
[0352] Takase, K., and Ebner, K. E. (1984) Interaction of
galactosyltransferase with R-lactalbumin and substrates, Curr. Top,
Cell Regul. 24, 51-62. [0353] Unligil, U. M. and Rini, J. M. (2000)
Glycosyltransferase structure and mechanism. Curr. Opin. Struct.
Biol. 10, 510-517.
Sequence CWU 1
1
81819DNAHomo sapiens 1ctgcccgcat gccctgagga gtccccgctg cttgtgggcc
ccatgctgat tgagtttaac 60atgcctgtgg acctggagct cgtggcaaag cagaacccaa
atgtgaagat gggcggccgc 120tatgccccca gggactgcgt ctctcctcac
aaggtggcca tcatcattcc attccgcaac 180cggcaggagc acctcaagta
ctggctatat tatttgcacc cagtcctgca gcgccagcag 240ctggactatg
gcatctatgt tatcaaccag gcgggagaca ctatattcaa tcgtgctaag
300ctcctcaatg ttggctttca agaagccttg aaggactatg actacacctg
ctttgtgttt 360agtgacgtgg acctcattcc aatgaatgac cataatgcgt
acaggtgttt ttcacagcca 420cggcacattt ccgttgcaat ggataagttt
ggattcagcc taccttatgt tcagttgttt 480ggaggtgtct ctgctctaag
taaacaacag tttctaacca tcaatggatt tcctaataat 540tattggggct
ggggaggaga agatgatgac atttttaaca gattagtttt tagaggcatg
600tctatatctc gcccaaatgc tgtggtcggg aggacgcgtc acatccgcca
ctcgagagac 660aagaaaaatg aacccaatcc tcagaggttt gaccgaattg
cacacacaaa ggagacaatg 720ctctctaatg gtttgaactc actcacctac
caggtgctgg atgtacagag atacccattg 780tatacccaaa tcacagtgga
catcgggaca ccgagctag 8192272PRTHomo sapiens 2Leu Pro Ala Cys Pro
Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu 1 5 10 15 Ile Glu Phe
Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn 20 25 30 Pro
Asn Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser 35 40
45 Pro His Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His
50 55 60 Leu Lys Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg
Gln Gln 65 70 75 80Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly
Asp Thr Ile Phe 85 90 95 Asn Arg Ala Lys Leu Leu Asn Val Gly Phe
Gln Glu Ala Leu Lys Asp 100 105 110 Tyr Asp Tyr Thr Cys Phe Val Phe
Ser Asp Val Asp Leu Ile Pro Met 115 120 125 Asn Asp His Asn Ala Tyr
Arg Cys Phe Ser Gln Pro Arg His Ile Ser 130 135 140 Val Ala Met Asp
Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Leu Phe 145 150 155 160Gly
Gly Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly 165 170
175 Phe Pro Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe
180 185 190 Asn Arg Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn
Ala Val 195 200 205 Val Gly Arg Thr Arg His Ile Arg His Ser Arg Asp
Lys Lys Asn Glu 210 215 220 Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala
His Thr Lys Glu Thr Met 225 230 235 240Leu Ser Asn Gly Leu Asn Ser
Leu Thr Tyr Gln Val Leu Asp Val Gln 245 250 255 Arg Tyr Pro Leu Tyr
Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 260 265 270 3415PRTHomo
sapiens 3Met Arg Leu Arg Glu Pro Leu Leu Ser Arg Ser Ala Ala Met
Pro Gly 1 5 10 15 Met Arg Phe Arg Glu Gln Phe Leu Gly Gly Ser Ala
Ala Met Pro Gly 20 25 30 Ala Thr Leu Gln Arg Ala Cys Arg Leu Leu
Val Ala Val Cys Ala Leu 35 40 45 His Leu Gly Val Thr Leu Val Tyr
Tyr Leu Ser Gly Arg Asp Leu Ser 50 55 60 Arg Leu Pro Gln Leu Val
Gly Val Ser Ser Thr Leu Gln Gly Gly Thr 65 70 75 80Asn Gly Ala Ala
Ala Ser Lys Gln Pro Pro Gly Glu Gln Arg Pro Arg 85 90 95 Gly Ala
Arg Pro Pro Pro Pro Leu Gly Val Ser Pro Lys Pro Arg Pro 100 105 110
Gly Leu Asp Ser Ser Pro Gly Ala Ala Ser Gly Pro Gly Leu Lys Ser 115
120 125 Asn Leu Ser Ser Leu Pro Val Pro Thr Thr Thr Gly Leu Leu Ser
Leu 130 135 140 Pro Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro
Met Leu Ile 145 150 155 160Asp Phe Asn Ile Ala Val Asp Leu Glu Leu
Leu Ala Lys Lys Asn Pro 165 170 175 Glu Ile Lys Thr Gly Gly Arg Tyr
Ser Pro Lys Asp Cys Val Ser Pro 180 185 190 His Lys Val Ala Ile Ile
Ile Pro Phe Arg Asn Arg Gln Glu His Leu 195 200 205 Lys Tyr Trp Leu
Tyr Tyr Leu His Pro Ile Leu Gln Arg Gln Gln Leu 210 215 220 Asp Tyr
Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Met Phe Asn 225 230 235
240Arg Ala Lys Leu Leu Asn Ile Gly Phe Gln Glu Ala Leu Lys Asp Tyr
245 250 255 Asp Tyr Asn Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro
Met Asp 260 265 270 Asp Arg Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg
His Ile Ser Val 275 280 285 Ala Met Asp Lys Phe Gly Phe Ser Leu Pro
Tyr Val Gln Tyr Phe Gly 290 295 300 Gly Val Ser Ala Leu Ser Lys Gln
Gln Pro Leu Ala Ile Asn Gly Phe 305 310 315 320Pro Asn Asn Tyr Trp
Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn 325 330 335 Arg Leu Val
His Lys Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val 340 345 350 Gly
Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro 355 360
365 Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Arg
370 375 380 Phe Asp Gly Leu Asn Ser Leu Thr Tyr Lys Val Leu Asp Val
Gln Arg 385 390 395 400Tyr Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile
Gly Thr Pro Arg 405 410 4154402PRTBos sp. 4Met Lys Phe Arg Glu Pro
Leu Leu Gly Gly Ser Ala Ala Met Pro Gly 1 5 10 15 Ala Ser Leu Gln
Arg Ala Cys Arg Leu Leu Val Ala Val Cys Ala Leu 20 25 30 His Leu
Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Arg 35 40 45
Arg Leu Pro Gln Leu Val Gly Val His Pro Pro Leu Gln Gly Ser Ser 50
55 60 His Gly Ala Ala Ala Ile Gly Gln Pro Ser Gly Glu Leu Arg Leu
Arg 65 70 75 80Gly Val Ala Pro Pro Pro Pro Leu Gln Asn Ser Ser Lys
Pro Arg Ser 85 90 95 Arg Ala Pro Ser Asn Leu Asp Ala Tyr Ser His
Pro Gly Pro Gly Pro 100 105 110 Gly Pro Gly Ser Asn Leu Thr Ser Ala
Pro Val Pro Ser Thr Thr Thr 115 120 125 Arg Ser Leu Thr Ala Cys Pro
Glu Glu Ser Pro Leu Leu Val Gly Pro 130 135 140 Met Leu Ile Glu Phe
Asn Ile Pro Val Asp Leu Lys Leu Ile Glu Gln 145 150 155 160Gln Asn
Pro Lys Val Lys Leu Gly Gly Arg Tyr Thr Pro Met Asp Cys 165 170 175
Ile Ser Pro His Lys Val Ala Ile Ile Ile Leu Phe Arg Asn Arg Gln 180
185 190 Glu His Leu Lys Tyr Trp Leu Tyr Tyr Leu His Pro Met Val Gln
Arg 195 200 205 Gln Gln Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala
Gly Glu Ser 210 215 220 Met Phe Asn Arg Ala Lys Leu Leu Asn Val Gly
Phe Lys Glu Ala Leu 225 230 235 240Lys Asp Tyr Asp Tyr Asn Cys Phe
Val Phe Ser Asp Val Asp Leu Ile 245 250 255 Pro Met Asn Asp His Asn
Thr Tyr Arg Cys Phe Ser Gln Pro Arg His 260 265 270 Ile Ser Val Ala
Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln 275 280 285 Tyr Phe
Gly Gly Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Ser Ile 290 295 300
Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp 305
310 315 320Ile Tyr Asn Arg Leu Ala Phe Arg Gly Met Ser Val Ser Arg
Pro Asn 325 330 335 Ala Val Ile Gly Lys Cys Arg Met Ile Arg His Ser
Arg Asp Lys Lys 340 345 350 Asn Glu Pro Asn Pro Gln Arg Phe Asp Arg
Ile Ala His Thr Lys Glu 355 360 365 Thr Met Leu Ser Asp Gly Leu Asn
Ser Leu Thr Tyr Met Val Leu Glu 370 375 380 Val Gln Arg Tyr Pro Leu
Tyr Thr Lys Ile Thr Val Asp Ile Gly Thr 385 390 395 400Pro Ser
52013DNAHomo sapiens 5ttggcctggc ctgcttgtcg ctgggatctg aatgaccaaa
ccacttccca ccatggctcc 60tggaaggact aaatgaagtc atgagtataa agtgctcctg
catggccagc agccggatgc 120ccgggcccac tgggcgggcc agtggccgcc
tgcgggatga gcagactgct gggggggacg 180ctggagcgcg tctgcaaggc
tgtgctcctt ctctgcctgc tgcacttcct cgtggccgtc 240atcctctact
ttgacgtcta cgcccagcac ctggccttct tcagccgctt cagtgcccga
300ggccctgccc atgccctcca cccagctgct agcagcagca gcagcagcag
caactgctcc 360cggcccaacg ccaccgcctc tagctccggg ctccctgagg
tccccagtgc cctgcccggt 420cccacggctc ccacgctgcc accctgtcct
gactcgccac ctggtcttgt gggcagactg 480ctgatcgagt tcacctcacc
catgcccctg gagcgggtgc agagggagaa cccaggcgtg 540ctcatgggcg
gccgatacac accgcccgac tgcaccccag cccagacggt ggcggtcatc
600atccccttta gacaccggga acaccacctg cgctactggc tccactatct
acaccccatc 660ttgaggcggc agcggctgcg ctacggcgtc tatgtcatca
accagcatgg tgaggacacc 720ttcaaccggg ccaagctgct taacgtgggc
ttcctagagg cgctgaagga ggatgccgcc 780tatgactgct tcatcttcag
cgatgtggac ctggtcccca tggatgaccg caacctatac 840cgctgcggcg
accaaccccg ccactttgcc attgccatgg acaagtttgg cttccggctt
900ccctatgctg gctactttgg aggtgtgtca ggcctgagta aggctcagtt
tctgagaatc 960aatggcttcc ccaatgagta ctggggctgg ggtggcgagg
atgatgacat cttcaaccgg 1020atctccctga ctgggatgaa gatctcacgc
ccagacatcc gaatcggccg ctaccgcatg 1080atcaagcacg accgcgacaa
gcataacgaa cctaaccctc agaggtttac caagattcaa 1140aacacgaagc
tgaccatgaa gcgggacggc attgggtcag tgcggtacca ggtcttggag
1200gtgtctcggc aaccactctt caccaatatc acagtggaca ttgggcggcc
tccgtcgtgg 1260ccccctcggg gctgacacta atggacagag gctctcggtg
ccgaagattg cctgccagag 1320gactgaccac agcctggctg gcagctgctc
tgtggaggac ctccaggact gagactgggc 1380tctgttttcc aagggtcttc
actaggcccc ctagctacac ctggaagttt cagaacccac 1440tttggggggc
ctcctgcctg ggcaggctct tcaagtgtgg ccctctttgg agtcaaccct
1500ccttcccgac cccctccccc tagcccagcc ccagtcactg tcagggtcgg
gccagcccct 1560gcactgcctc gcagagtggc ctgggctagg tcactccacc
tctctgtgcc tcagtttccc 1620ccccttgagt cccctagggc ctggaagggt
gggaggtatg tctagggggc agtgtctctt 1680ccagggggaa ttctcagctc
ttgggaaccc ccttgctccc aggggagggg aaaccttttt 1740cattcaacat
tgtagggggc aagctttggt gcgccccctg ctgaggagca gccccaggag
1800gggaccagag gggatgctgt gtcgctgcct gggatcttgg ggttggcctt
tgcatgggag 1860gcaggtgggg cttggatcag taagtctggt tcccgcctcc
ctgtctgaga gaggaggcag 1920gagccccagg gccggcttgt gtttgtacat
tgcacagaaa cttgtgtggg tgctttagta 1980aaaaacgtga atggaaaaaa
aaaaaaaaaa aaa 20136372PRTHomo sapiens 6Met Ser Arg Leu Leu Gly Gly
Thr Leu Glu Arg Val Cys Lys Ala Val 1 5 10 15 Leu Leu Leu Cys Leu
Leu His Phe Leu Val Ala Val Ile Leu Tyr Phe 20 25 30 Asp Val Tyr
Ala Gln His Leu Ala Phe Phe Ser Arg Phe Ser Ala Arg 35 40 45 Gly
Pro Ala His Ala Leu His Pro Ala Ala Ser Ser Ser Ser Ser Ser 50 55
60 Ser Asn Cys Ser Arg Pro Asn Ala Thr Ala Ser Ser Ser Gly Leu Pro
65 70 75 80Glu Val Pro Ser Ala Leu Pro Gly Pro Thr Ala Pro Thr Leu
Pro Pro 85 90 95 Cys Pro Asp Ser Pro Pro Gly Leu Val Gly Arg Leu
Leu Ile Glu Phe 100 105 110 Thr Ser Pro Met Pro Leu Glu Arg Val Gln
Arg Glu Asn Pro Gly Val 115 120 125 Leu Met Gly Gly Arg Tyr Thr Pro
Pro Asp Cys Thr Pro Ala Gln Thr 130 135 140 Val Ala Val Ile Ile Pro
Phe Arg His Arg Glu His His Leu Arg Tyr 145 150 155 160Trp Leu His
Tyr Leu His Pro Ile Leu Arg Arg Gln Arg Leu Arg Tyr 165 170 175 Gly
Val Tyr Val Ile Asn Gln His Gly Glu Asp Thr Phe Asn Arg Ala 180 185
190 Lys Leu Leu Asn Val Gly Phe Leu Glu Ala Leu Lys Glu Asp Ala Ala
195 200 205 Tyr Asp Cys Phe Ile Phe Ser Asp Val Asp Leu Val Pro Met
Asp Asp 210 215 220 Arg Asn Leu Tyr Arg Cys Gly Asp Gln Pro Arg His
Phe Ala Ile Ala 225 230 235 240Met Asp Lys Phe Gly Phe Arg Leu Pro
Tyr Ala Gly Tyr Phe Gly Gly 245 250 255 Val Ser Gly Leu Ser Lys Ala
Gln Phe Leu Arg Ile Asn Gly Phe Pro 260 265 270 Asn Glu Tyr Trp Gly
Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg 275 280 285 Ile Ser Leu
Thr Gly Met Lys Ile Ser Arg Pro Asp Ile Arg Ile Gly 290 295 300 Arg
Tyr Arg Met Ile Lys His Asp Arg Asp Lys His Asn Glu Pro Asn 305 310
315 320Pro Gln Arg Phe Thr Lys Ile Gln Asn Thr Lys Leu Thr Met Lys
Arg 325 330 335 Asp Gly Ile Gly Ser Val Arg Tyr Gln Val Leu Glu Val
Ser Arg Gln 340 345 350 Pro Leu Phe Thr Asn Ile Thr Val Asp Ile Gly
Arg Pro Pro Ser Trp 355 360 365 Pro Pro Arg Gly 370 74PRTHomo
sapiensMOD_RES(1)Arg-[GlcNAcbeta1,2-Manalpha1, 6-(GlcNAcbeta1,2-Man
alpha1,3)-Manbeta1,4-GlcNAcbeta1,4-GlcNAcbeta] 7Arg Asn Glu Gly 1
813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Pro Thr Thr Asp Ser Thr Thr Pro Ala Pro Thr Thr
Lys 1 5 10
* * * * *