U.S. patent application number 11/217544 was filed with the patent office on 2007-11-22 for nucleic acids encoding a chimeric glycosyltransferase.
This patent application is currently assigned to Austin Research Institute. Invention is credited to Ian Farquhar Campbell McKenzie, Mauro Sergio Sandrin.
Application Number | 20070271621 11/217544 |
Document ID | / |
Family ID | 25645233 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070271621 |
Kind Code |
A1 |
McKenzie; Ian Farquhar Campbell ;
et al. |
November 22, 2007 |
Nucleic acids encoding a chimeric glycosyltransferase
Abstract
The invention relates to nucleic acids which encode
glycosyltransferase and are useful in producing cells and organs
from one species which may be used for transplantation into a
recipient of another species. It also relates to the production of
nucleic acids which, when present in cells of a transplanted organ,
result in reduced levels of antibody recognition of the
transplanted organ.
Inventors: |
McKenzie; Ian Farquhar
Campbell; (Brunswick, AU) ; Sandrin; Mauro
Sergio; (Brunswick, AU) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Austin Research Institute
Heidelberg
AU
|
Family ID: |
25645233 |
Appl. No.: |
11/217544 |
Filed: |
September 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09051034 |
Mar 31, 1998 |
7001998 |
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PCT/AU97/00492 |
Aug 1, 1997 |
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11217544 |
Sep 1, 2005 |
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60024279 |
Aug 21, 1996 |
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Current U.S.
Class: |
800/13 ;
435/320.1; 435/325; 435/375; 435/440; 435/476; 536/23.2;
536/25.3 |
Current CPC
Class: |
C12N 9/1048 20130101;
C07K 2319/00 20130101; A61K 38/00 20130101; A61K 48/00
20130101 |
Class at
Publication: |
800/013 ;
435/320.1; 435/325; 435/375; 435/440; 435/476; 536/023.2;
536/025.3 |
International
Class: |
C12N 5/10 20060101
C12N005/10; A01K 67/033 20060101 A01K067/033; C07H 21/00 20060101
C07H021/00; C12N 15/63 20060101 C12N015/63; C12N 15/74 20060101
C12N015/74; C12N 5/02 20060101 C12N005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 2, 1996 |
AU |
PO 1402 |
Claims
1. A nucleic acid encoding a chimeric enzyme, wherein said chimeric
enzyme comprises a catalytic domain of a first glycosyltransferase
and a localisation signal of a second glycosyltransferase, whereby
when said nucleic acid is expressed in a cell said chimeric enzyme
is located in an area of the cell where it is able to compete for
substrate with a second glycosyltransferase, resulting in reduced
levels of a product from said second glycosyltransferase.
2. A nucleic acid according to claim 1, wherein said localisation
signal localises said catalytic domain thereby to enable the
catalytic domain to compete with said second glycosyltransferase
for a substrate.
3. A nucleic acid according to claim 1, wherein the localisation
signal is derived from a glycosyltransferase which produces
glycosylation patterns which are recognised as foreign by a
transplant recipient.
4. A nucleic acid according to any one claim 1, wherein the
localisation signal comprises the amino terminus of the second
glycosyltransferase.
5. A nucleic acid according to claim 1, wherein the localisation
signal is derived from .alpha.(1,3) -galactosyltransferase.
6. A nucleic acid according to claim 1, wherein the first
glycosyltransferase is selected from the group consisting of
H-transferase, secretor sialyltransferase, a galactosyl sulphating
enzyme or a phosphorylating enzyme.
7. A nucleic acid according to claim 1, wherein the catalytic
domain and the localisation signal each originates from a mammal
selected from the group consisting of human, primates, ungulates,
dogs, mice, rats and rabbits.
8. A nucleic acid according to claim 1, wherein the localisation
signal is derived from the same species as the cell which the
nucleic acid is intended to transform.
9. A nucleic acid according to claim 1, comprising a sequence
encoding the catalytic domain of H transferase and a nucleic acid
sequence encoding a localisation signal from Gal transferase.
10. A nucleic acid according to claim 9, wherein the catalytic
domain and the localisation signal are derived from pigs.
11. A nucleic acid according to claim 1, which encodes gtHT as
defined herein.
12. A vehicle comprising a nucleic acid according to claim 1.
13. A vehicle according to claim 12, selected from the group
consisting of an expression vector, plasmid and phage.
14. A vehicle according to claim 12 which enables said nucleic acid
to be expressed in prokaryotes or in eukaryotes.
15. An isolated nucleic acid molecule encoding a localisation
signal of a glycosyltransferase.
16. An isolated nucleic acid molecule according to claim 15,
wherein the signal encoded comprises an amino terminus of
gal-transferase.
17. A method of producing a nucleic acid according to claim 1,
comprising the step of operably linking a nucleic acid sequence
encoding a catalytic domain from a first glycosyltransferase to a
nucleic acid sequence encoding a localisation signal of a second
glycosyltransferase.
18. A method of reducing the level of a carbohydrate exhibited on
the surface of a cell, said method comprising causing a nucleic
acid to be expressed in said cell wherein said nucleic acid encodes
a chimeric enzyme which comprises a catalytic domain of a first
glycosyltransferase and a localisation signal of a second
glycosyltransferase, whereby said chimeric enzyme is located in an
area of the cell where it is able to compete for substrate with
said second glycosyltransferase, and wherein said second
glycosyltransferase is capable of producing said carbohydrate.
19. A method of producing a cell from a donor species which in
immunologically acceptable to a recipient species by reducing
levels of carbohydrate on said cell which cause it to be recognised
as non-self by the recipient, said method comprising causing a
nucleic acid to be expressed in said cell wherein said nucleic acid
encodes a chimeric enzyme which comprises a catalytic domain of a
first glycosyltransferase and a localisation signal of a second
glycosyltransferase, whereby said chimeric enzyme is located in an
area of the cell where it is able to compete for substrate with
said second glycosyltransferase, and wherein said second
glycosyltransferase is capable of producing said carbohydrate.
20. A cell produced by a method according to claim 19.
21. An organ comprising a cell according to claim 20.
22. A non-human transgenic animal, organ or cell comprising the
nucleic acid according to claim 1.
23. An expression unit which expresses a nucleic acid according to
claim 1, resulting in a cell which is immunologically acceptable to
an animal having reduced levels of a carbohydrate on its surface,
which carbohydrate is recognised as non-self by said species.
24. An expression unit according to claim 23, selected from the
group consisting of a retroviral packaging cassette, retroviral
construct or retroviral producer cell.
25. A method of producing an expression unit according to claim 23,
said unit having reduced levels of a carbohydrate on its surface
wherein the carbohydrate is recognised as non-self by a species,
comprising transforming/transfecting a retroviral packaging cell or
a retroviral producer cell with the nucleic acid of the invention
under conditions such that the chimeric enzyme is produced.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to nucleic acids which encode
glycosyltransferase and are useful in producing cells and organs
from one species which may be used for transplantation into a
recipient of another species. Specifically the invention concerns
production of nucleic acids which, when present in cells of a
transplanted organ, result in reduced levels of antibody
recognition of the transplanted organ.
BACKGROUND OF THE INVENTION
[0002] The transplantation of organs is now practicable, due to
major advances in surgical and other techniques. However,
availability of suitable human organs for transplantation is a
significant problem. Demand outstrips supply. This has caused
researchers to investigate the possibility of using non-human
organs for transplantation.
[0003] Xenotransplantation is the transplantation of organs from
one species to a recipient of a different species. Rejection of the
transplant in such cases is a particular problem, especially where
the donor species is more distantly related, such as donor organs
from pigs and sheep to human recipients. Vascular organs present a
special difficulty because of hyperacute rejection (HAR).
[0004] HAR occurs when the complement cascade in the recipient is
initiated by binding of antibodies to donor endothelial cells.
[0005] Previous attempts to prevent HAR have focused on two
strategies: modifying the immune system of the host by inhibition
of systemic complement formation (1,2), and antibody depletion
(3,4). Both strategies have been shown to prolong xenograft
survival temporarily. However, these methodologies are
therapeutically unattractive in that they are clinically
impractical, and would require chronic immunosuppressive
treatments. Therefore, recent efforts to inhibit HAR have focused
on genetically modifying the donor xenograft. One such strategy has
been to achieve high-level expression of species-restricted human
complement inhibitory proteins in vascularized pig organs via
transgenic engineering (5-7). This strategy has proven to be useful
in that it has resulted in the prolonged survival of porcine
tissues following antibody and serum challenge (5,6). Although
increased survival of the transgenic tissues was observed,
long-term graft survival was not achieved (6). As observed in these
experiments and also with systemic complement depletion, organ
failure appears to be related to an acute antibody-dependent
vasculitis. (1,5).
[0006] In addition to strategies aimed at blocking complement
activation on the vascular endothelial cell surface of the
xenograft, recent attention has focused on identification of the
predominant xenogeneic epitope recognized by high-titre human
natural antibodies. It is now accepted that the terminal galactosyl
residue, Gal-.alpha.-(1,3)-Gal, is the dominant xenogeneic epitope
(8-15). This epitope is absent in Old World primates and humans
because the .alpha.-(1,3)-galactosyltransferase (gal-transferase or
GT) is non-functional in these species. DNA sequence comparison of
the human gene to .alpha.-(1,3)-galactosyltransferase genes from
the mouse (16,17), ox (18), and pig (12) revealed that the human
gene contained two frameshift mutations, resulting in a
non-functional pseudogene (20,21). Consequently, humans and Old
World primates have pre-existing high-titre antibodies directed at
this Gal-.alpha.(1,3)-Gal moiety as the dominant xenogeneic
epitope.
[0007] One strategy developed was effective to stably reduce the
expression of the predominant Gal-.alpha.(1,3)-Gal epitope. This
strategy took advantage of an intracellular competition between the
gal-transferase and .alpha.(1,2)-fucosyltransferase (H-transferase)
for a common acceptor substrate. The gal-transferase catalyzes the
transfer of a terminal galactose moiety to an N-acetyl lactosamine
acceptor substrate, resulting in the formation of the terminal
Gal-.alpha.(1,3)-Gal epitope. Conversely, H-transferase catalyzes
the transfer of a fucosyl residue to the N-acetyl lactosamine
acceptor substrate, and generates a fucosylated N-acetyl
lactosamine (H-antigen, i.e., the O blood group antigen), a
glycosidic structure that is universally tolerated. Although it was
reported that expression of human H-transferase transfected cells
resulted in high level expression of the non-antigenic H-epitope
and significantly reduced the expression of the
Gal-.alpha.(1,3)-Gal xenoepitope, there are still significant
levels of Gal.alpha.(1,3)-Gal epitope present on such cells.
SUMMARY OF THE INVENTION
[0008] In view of the foregoing, it is an object of the present
invention to further reduce levels of undesirable epitopes in
cells, tissues and organs which may be used in transplantation.
[0009] In work leading up to the invention the inventors
surprisingly discovered that the activity of H transferase may be
further increased by making a nucleic acid which encodes a H
transferase catalytic domain but is anchored in the cell at a
location where it is better able to compete for substrate with gal
transferase. Although work by the inventors focused on a chimeric H
transferase, other glycosyltransferase enzymes may also be produced
in accordance with the invention.
[0010] Accordingly, in a first aspect the invention provides a
nucleic acid encoding a chimeric enzyme, wherein said chimeric
enzyme comprises a catalytic domain of a first glycosyltransferase
and a localization signal of a second glycosyltransferase, whereby
when said nucleic acid is expressed in a cell said chimeric enzyme
is located in an area of the cell where it is able to compete for
substrate with a second glycosyltransferase, resulting in reduced
levels of a product from said second glycosyltransferase.
[0011] Preferably the nucleic acid is in an isolated form; that is
the nucleic acid is at least partly purified from other nucleic
acids or proteins.
[0012] Preferably the nucleic acid comprises the correct sequences
for expression, more preferably for expression in a eukaryotic
cell. The nucleic acid may be present on any suitable eukaryotic
expression vector such as pcDNA (Invitrogen). The nucleic acid may
also be present on other vehicles whether suitable for eukaryotes
or not, such as plasmids, phages and the like.
[0013] Preferably the catalytic domain of the first
glycosyltransferase is derived from H transferase, secretor
sialyltransferase, a galactosyl sulphating enzyme or a
phosphorylating enzyme.
[0014] The nucleic acid sequence encoding the catalytic domain may
be derived from, or similar to a glycosyltransferase from any
species. Preferably said species is a mammalian species such as
human or other primate species, including Old World monkeys, or
other mammals such as ungulates (for example pigs, sheep, goats,
cows, horses, deer, camels) or dogs, mice, rats and rabbits. The
term "similar to" means that the nucleic acid is at least partly
homologous to the glycocyltransferase genes described above. The
term also extends to fragments of and mutants, variants and
derivatives of the catalytic domain whether naturally occurring or
man made.
[0015] Preferably the localization signal is derived from a
glycosyltransferase which produces glycosylation patterns which are
recognized as foreign by a transplant recipient. More preferably
the localization signal is derived from .alpha.(1,3)
galactosyltransferase. The effect of this is to downregulate the
level of Gal-.alpha.(1,3)-Gal produced in a cell when the nucleic
acid is expressed by the cell.
[0016] The nucleic acid sequence encoding the localization signal
may be derived from any species such as those described above.
Preferably it is derived from the same species as the cell which
the nucleic acid is intended to transform i.e., if pig cells are to
be transformed, preferably the localization signal is derived from
pig.
[0017] More preferably the nucleic acid comprises a nucleic acid
sequence encoding the catalytic domain of H transferase and a
nucleic acid sequence encoding a localization signal from Gal
transferase. Still more preferably both nucleic acid sequences are
derived from pigs. Even more preferably the nucleic acid encodes
gtHT described herein.
[0018] The term "nucleic acid" refers to any nucleic acid
comprising natural or synthetic purines and pyrimidines. The
nucleic acid may be DNA or RNA, single or double stranded or
covalently closed circular.
[0019] The term "catalytic domain" of the chimeric enzyme refers to
the amino acid sequences necessary for the enzyme to function
catalytically. This comprises one or more contiguous or
non-contiguous amino acid sequences. Other non-catalytically active
portions also may be included in the chimeric enzyme.
[0020] The term "glycosyltransferase" refers to a polypeptide with
an ability to move carbohydrates from one molecule to another.
[0021] The term "derived from" means that the catalytic domain is
based on, or is similar, to that of a native enzyme. The nucleic
acid sequence encoding the catalytic domain is not necessarily
directly derived from the native gene. The nucleic acid sequence
may be made by polymerase chain reaction (PCR), constructed de novo
or cloned.
[0022] The term "localization signal" refers to the amino acid
sequence of a glycosyltransferase which is responsible for
anchoring it in location within the cell. Generally localization
signals comprise amino terminal "tails" of the enzyme. The
localization signals are derived from a second glycosyltransferase,
the activity of which it is desired to minimise. The localization
of a catalytic domain of a first enzyme in the same area as the
second glycosyltransferase means that the substrate reaching that
area is likely to be acted on by the catalytic domain of the first
enzyme, enabling the amount of substrate catalysed by the second
enzyme to be reduced.
[0023] The term "area of the cell" refers to a reglon, compartment
or organelle of the cell. Preferably the area of the cell is a
secretory organelle such as the Golgi apparatus.
[0024] In another aspect the invention provides an isolated nucleic
acid molecule encoding a localization signal of a
glycosyltransferase. Preferably the signal encoded comprises an
amino terminus of said molecule; more preferably it is the amino
terminus of gal transferase. The gal transferase may be derived
from or based on a gal transferase from any mammalian species, such
as those described above. Particularly preferred sequences are
those derived from pig, mouse or cattle.
[0025] In another aspect the invention relates to a method of
producing a nucleic acid encoding a chimeric enzyme, said enzyme
comprising a catalytic domain of a first glycosyltransferase and a
localization signal of a second glycosyltransferase whereby when
said nucleic acid is expressed in a cell said chimeric enzyme is
located in an area of the cell where it is able to compete for
substrate with a second glycosyltransferase said method comprising
operably linking a nucleic acid sequence encoding a catalytic
domain from a first glycosyltransferase to a nucleic acid sequence
encoding a localization signal of a second glycosyltransterase.
[0026] The term "operably linking" means that the nucleic acid
sequences are ligated such that a functional protein is able to be
transcribed and translated.
[0027] Those skilled in the art will be aware of various techniques
for producing the nucleic acid. Standard techniques such as those
described in Sambrook et al may be employed.
[0028] Preferably the nucleic acid sequences are the preferred
sequences described above.
[0029] In another aspect the invention provides a method of
reducing the level of a carbohydrate exhibited on the surface of a
cell, said method comprising causing a nucleic acid to be expressed
in said cell wherein said nucleic acid encodes a chimeric enzyme
which comprises a catalytic domain of a first glycosyltransferase
and a localization signal of a second glycosyltransferase, whereby
said chimeric enzyme is located in an area of the cell where it is
able to compete for substrate with said second glycosyltransferase,
and wherein said second glycosyltransferase is capable of producing
said carbohydrate.
[0030] The term "reducing the level of a carbohydrate" refers to
lowering, minimising, or in some cases, ablating the amount of
carbohydrate displayed on the surface of the cell. Preferably said
carbohydrate is capable of stimulating recognition of the cell as
"non-self" by the immune system of an animal. The reduction of such
a carbohydrate therefore renders the cell, or an organ composed of
said cells, more acceptable to the immune system of a recipient
animal in a transplant situation or gene therapy situation.
[0031] The term "causing a nucleic acid to be expressed" means that
the nucleic acid is introduced into the cell (i.e. by
transformation/transfection or other suitable means) and contains
appropriate signals to allow expression in the cells.
[0032] The cell may be any suitable cell, preferably mammalian,
such as that of a New World monkey, ungulate (pig, sheep, goat,
cow, horse, deer, camel, etc.) or other species such as dogs.
[0033] In another aspect the invention provides a method of
producing a cell from one species (the donor) which is
immunologically acceptable to another species (the recipient) by
reducing levels of carbohydrate on said cell which cause it to be
recognized as non-self by the other species, said method comprising
causing a nucleic acid to be expressed in said cell wherein said
nucleic acid encodes a chimeric enzyme which comprises a catalytic
domain of a first glycosyltransferase and a localization signal of
a second glycosyltransferase, whereby said chimeric enzyme is
located in an area of the cell where it is able to compete for
substrate with said second glycosyltransferase, and wherein said
second glycosyltransferase is capable of producing said
carbohydrate.
[0034] The term "immunologically acceptable" refers to producing a
cell, or an organ made up of numbers of the cell. which does not
cause the same degree of immunological reaction in the recipient
species as a native cell from the donor species. Thus the cell may
cause a lessened immunological reaction, only requiring low levels
of immunosuppressive therapy to maintain such a transplanted organ
or no immunosuppression therapy.
[0035] The cell may be from any of the species mentioned above.
Preferably the cell is from a New World primate or a pig. More
preferably the cell is from a pig.
[0036] The invention extends to cells produced by the above method
and also to organs comprising the cells.
[0037] The invention further extends to non-human transgenic
animals harbouring the nucleic acid of the invention. Preferably
the species is a human, ape or Old World monkey.
[0038] The invention also extends to the proteins produced by the
nucleic acid. Preferably the proteins are in an isolated form.
[0039] In another aspect the invention provides an expression unit
which expresses the nucleic acid of the invention, resulting in a
cell which is immunologically acceptable to an animal having
reduced levels of a carbohydrate on its surface, which carbohydrate
is recognized as non-self by said species. In a preferred
embodiment, the expression unit is a retroviral packaging cell,
cassette, a retroviral construct or retroviral producer cell.
[0040] Preferably the species is a human, ape or old World
monkey.
[0041] The retroviral packaging cells or retroviral producer cells
may be cells of any animal origin where it is desired to reduce the
level of carbohydrates on its surface to make it more
immunologically acceptable to a host. Such cells may be derived
from mammals such as canine, rodent or ruminant species and the
like.
[0042] The retroviral packaging and/or producer cells may be used
in applications such as gene therapy. General methods involving use
of such cells are described in PCT/US95107554 and the references
discussed therein.
[0043] The invention also extends to a method of producing a
retroviral packaging cell or a retroviral producer cell having
reduced levels of a carbohydrate on its surface wherein the
carbohydrate is recognized as nonself by a species, comprising
transforming/transfecting a retroviral packaging cell or a
retroviral producer cell with the nucleic acid of the invention
under conditions such that the chimeric enzyme is produced.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 Schematic diagram of normal and chimeric
glycosyltransferases
[0045] The diagram shows normal glycosyltransferases porcine
.alpha.(1,3)galactosyltransferase (GT) and human
.alpha.(1,2)fucosyltransferase (HT), and chimeric transferases
ht-GT in which the cytoplasmic domain of GT has been completely
replaced by the cytoplasmic domain of HT, and gt-HT in which the
cytoplasmic domain of HT has been entirely replaced by the
cytoplasmic domain of GT. The protein domains depicted are
cytoplasmic domain CYTO, transmembrane domain TM, stem region STEM,
catalytic domain CATALYTIC. The numbers refer to the amino acid
sequence of the corresponding normal transferase.
[0046] FIG. 2 Cell surface staining of COS cells transfected with
normal and chimeric transferases
[0047] Cells were transfected with normal GT or HT or with chimeric
transferases gt-HT or ht-GT and 48h later were stained with
FITC-labelled lectin IB4 or UEA1. Positive-staining cells were
visualized and counted by fluorescence microscopy. Results are from
at least three replicates and values are .+-.SEM.
[0048] FIG. 3. RNA analysis of transfected COS cells
[0049] Northern blots were performed on total RNA prepared from COS
cells transfected: Mock, mock-transfected; GT,transfected with
wild-type GT; GT1-6/HT, transfected with chimeric transferase
gt-HT; GT1-6/HT+HT1-8/GT, co-transfected with both chimeric
transferases gt-HT and ht-GT; HT1-8/GT, transfected with chimeric
transferase ht-GT; HT, transfected with normal HT; GT+HT,
co-transfected with both normal transferases GT and HT. Blots were
probed with a cDNA encoding GT (Top panel), HT (Middle panel) or
g-actin (Bottom panel).
[0050] FIG. 4. Enzyme kinetics of normal and chimeric
glycosyltransferases
[0051] Lineweaver-Burk plots for .alpha.(1,3) galactosyltransferase
(.quadrature.) and .alpha.(1,2) fucosyltransferaLse (.box-solid.)
to determine the apparent Km values for N-acetyl lactosamine.
Experiments were performed in triplicate, plots shown are of mean
values of enzyme activity of wild-type transferases, GT and HT, and
chimeric proteins ht-GT and gt-HT in transfected COS cell extracts
using phenyl-B-D Gal and N-acetyl lactosamine as acceptor
substrates.
[0052] FIG. 5. Staining of cells co-transfected with chimeric
transferases
[0053] Cells were co-transfected with cDNAs encoding normal
transferases GT+HT (panels A, B), with chimeric transferases
gt-HT+ht-GT (panels C, D), with HT+ht-GT (panels E, F) or with
GT+gt-HT (panels G, H) and 48h later were stained with
FITC-labelled lectin IB4 (panels A, C, E, G) or UEAI (panels B, D,
F, H).
[0054] FIG. 6 (SEQ ID Nos. 1 and 2) is a representation of the
nucleic acid sequence and corresponding amino acid sequence of pig
secretor.
[0055] FIG. 7 (SEQ ID Nos. 3 and 4) is a representation of the
nucleic acid sequence and corresponding amino acid sequence of pig
H.
[0056] FIG. 8 Cell surface staining of pig endothelial cell line
(PIEC) transfected with chimeric .alpha.(1,2)fucosyltransferase.
Cells were transfected and clones exhibiting stable integration
were stained with UEAI lectin and visualized by fluorescence
microscopy.
[0057] FIG. 9 screening of chimeric .alpha.(1,2)-fucosyltransferase
transferase in mice. Mice were injected with chimeric
a(1,2)-fucosyltransferase and the presence of the transferase was
analysed by dot blots.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0058] The nucleic acid sequences encoding the catalytic domain of
a glycosyltransferase may be any nucleic acid sequence such as
those described in PCT/US95/07554, which is herein incorporated by
reference, provided that it encodes a functional catalytic domain
with the desired glycosyltransferase activity.
[0059] Preferred catalytic domains from glycosyltransferase include
H transferase and secretor. Preferably these are based on human or
porcine sequences.
[0060] The nucleic acid sequences encoding the localization signal
of a second transglycosylase may be any nucleic acid sequence
encoding a signal sequence such as signal sequences disclosed in P
A Gleeson, R D Teasdale & J Bourke, Targeting of proteins to
the Golgi apparatus. Glycoconjugate J. (1994) 11: 381-394.
Preferably the localization signal is specific for the Golgi
apparatus, more preferably for that of the trans Golgi. Still more
preferably the localization signal is based on that of Gal
transferase. Even more preferably the localization signal is based
on porcine, murine or bovine sequences. Even more preferably the
nucleic acid encodes a signal sequence with following amino acid
sequence (in single letter code): MNVKGR (porcine) (SEQ ID No. 11),
MNVKGK (mouse) (SEQ ID No. 12), or MVVKGK (bovine) (SEQ ID No.
13).
[0061] Vectors for expression of the chimeric enzyme may be any
suitable vector, including those disclosed in PCT/US95/07554.
[0062] The nucleic acid of the invention can be used to produce
cells and organs with the desired glycosylation pattern by standard
techniques, such as those disclosed in PCT/US95107554. For example,
embryos may be transfected by standard techniques such as
microinjection of the nucleic acid in a linear form into the embryo
(22). The embryos are then used to produce live animals, the organs
of which may be subsequently used as donor organs for
implantation.
[0063] Cells, tissues and organs suitable for use in the invention
will generally be mammalian cells. Examples of suitable cells and
tissues such as endothelial cells, hepatic cells, pancreatic cells
and the like are provided in PCT/US95/07554.
[0064] The invention will now be described with reference to the
following non-limiting Examples.
[0065] Abbreviations
[0066] The abbreviations used are bp, base pair(s); FITC,
fluorescein isothiocyanate; GT, galactosyltransferase; H substance,
.alpha.(1,2)fucosyl lactosamine; HT,
.alpha.(1,2)fucosyltransferase; PCR, polymerase chain reaction;
Example 1 Cytoplasmic domains of glycosyltransferases play a
central role in the temporal action of enzymes
[0067] Experimental Procedures
EXAMPLE 1
Plasmids
[0068] The plasmids used were prepared using standard techniques
(7); pGT encodes the cDNA for the porcine
a(1,3)galactosyltransferase (23), pHT encodes the cDNA for the
a(1,2)fucosyltransferase (human) (25). Chimeric glycosyltransferase
cDNAs were generated by polymerase chain reaction as follows: an
1105 bp product ht-GT was generated using primers corresponding to
the 5' end of ht-GT (S'-GCGGATCCATGTGGCTCCGGAGCC
ATCGTCAGGTGGTTCTGTCAATGC TGCTTG-3') (SEQ ID No. 5) coding for
nucleotides 1-24 of HT (25) followed immediately by nucleotides
68-89 of GT (8) and containing a BamH1 site (double underlined) and
a primer corresponding to the 3' end of ht-GT
(5'-GCTCTAGAGCGTCAGATGTTATT TCTAACCAAATTATAC-3') (SEQ ID No. 6)
containing complementarity to nucleotides 1102-1127 of GT with an
Xbal site downstream of the translational stop site (double
underlined); an 1110 bp product gt-HT was generated using primers
corresponding to the 5' end of gt-HT
(5'-GCGGATCCATGAATGTCAAAGGAAGACTCTGCCTGGCCT TCCTGC-3') (SEQ ID No.
7) coding for nucleotides 49-67 of GT followed immediately by
nucleotides 25-43 of HT and containing a BamH1 site (double
underlined) and a primer corresponding to the 3' end of gt-HT
(5'-GCTCTAGAGCCTCAAGGCTTAG CCAATGTCCAGAG-3') (SEQ ID No. 8)
containing complementarity to nucleotides 1075-1099 of HT with a
Xbal site downstream of the translational stop site (double
underlined). PCR products were restricted BamH1/Xbal, gel-purified
and ligated into a BamH1/Xbal digested pcDNA1 expression vector
(Invitrogen) and resulted in two plasmids, pht-GT (encoding the
chimeric glycosyltransferase ht-GT) and pgt-HT (encoding the
chimeric glycosyltransferase gt-HT) which were characterized by
restriction mapping, Southern blotting and DNA sequencing.
[0069] Transfection and Serology--COS cells were maintained in
Dulbecco's modified Eagles Medium (DMEM) (Trace Biosciences Pty.
Ltd. , Castle Hill, NSW, Australia) and were transfected (1-10 pg
DNA/5.times.105 cells) using DEAE-Dextran (26); 48h later cells
were examined for cell surface expression of H substance or
Gal-.alpha.(1,3)-Gal using FITC-conjugated lectins: IB4 lectin
isolated from Griffonia simplicifolia (Sigma, St. Louis, Mo.)
detects Gal-.alpha.(1,3)-Gal (27); UEAI lectin isolated from Ulex
europaeus (Sigma, St. Louis, Mo.) detects H substance (28). H
substance was also detected by indirect immunofluorescence using a
monoclonal antibody (mAb) specific for the H substance (ASH-1952)
developed at the Austin Research Institute, using FITC-conjugated
goat antimouse IgG (Zymed Laboratories, San Francisco, Calif.) to
detect mAb binding. Fluorescence was detected by microscopy.
[0070] RNA Analyses--Cytoplasmic RNA was prepared from transfected
COS cells using RNAzol (Biotecx Laboratories, Houston, Tex.), and
total RNA was electrophoresed in a 1% agarose gel containing
formaldehyde, the gel blotted onto a nylon membrane and probed with
random primed GT or HT cDNA.
[0071] Glycosyltransferase assays--Forty-eight hours after
transfection, cells were washed twice with phosphate buffered
saline and lysed in 1% Triton X-100/100 mM cacodylate pH 6.5/25 mM
MnC12, at 4.degree. C. for 30 min; lysates were centrifuged and the
supernatant collected and stored at -70.degree. C. Protein
concentration was determined by the Bradford method using bovine
serum albumin as standard (29). Assays for HT activity (30) were
performed in 25 .mu.l containing 3mM [GDP-.sup.14C]fucose (specific
activity 287 mCi/mmol, Amersham International), 5 mM ATP, 5 OmM
MOPS pH 6.5, 20 mM MnC12, using 2-10 .mu.l of cell extract
(approximately 15-20 .mu.g of protein) and a range of
concentrations (7.5-75 mM) of the acceptor phenyl-B-D-galactoside
(Sigma). Samples were incubated for 2 h at 37.degree. C. and
reactions terminated by the addition of ethanol and water. The
amount of 'C.-fucose incorporated was counted after separation from
unincorporated label using Sep-Pak C18 cartridges
(Waters-millipore, Millford, Mass.). GT assays (31) were performed
in a volume of 25 .mu.l using 3mM UDP [.sup.3H]-Gal (specific
activity 189 mCi/mmol, Amersham International), 5 mM ATP, 100 mM
cacodylate pH 6.5, 20 mM MnC12 and various concentrations (1-10 mM)
of the acceptor N-acetyl lactosamine (Sigma). Samples were
incubated for 2 h at 37.degree. C. and the reactions terminated by
the addition of ethanol and water. .sup.3H-Gal incorporation was
counted after separation from non-incorporated UDP [.sup.3H]-Gal
using Dowex I anion exchange columns (BDH Ltd. , Poole, UK) or
Sep-Pak Accell plus QMA anion exchange cartridges
(Waters-Millipore, Millford, Mass.). All assays were performed in
duplicate and additional reactions were performed in the absence of
added acceptor molecules, to allow for the calculation of specific
incorporation of radioactivity.
[0072] Results
[0073] Expression of Chimeric
.alpha.(1,3)galactosyltransferase and .alpha.(1,2)
fucosyltransferase cDNAs
[0074] We had previously shown that when cDNAs encoding
.alpha.(1,3)galactosyltransferase (GT) and
.alpha.(1,2)fucosyltransferase (HT) were transfected separately
they could both function efficiently leading to expression of the
appropriate carbohydrates: Gal-.alpha.(1,3)-Gal for GT and H
substance for HT (32). However when the cDNAs for GT and HT were
transfected together, the HT appeared to "dominate" over the GT in
that H substance expression was normal, but Gal-.alpha.(1,3)-Gal
was reduced. We excluded trivial reasons for this effect and
considered that the localization of the enzymes may be the reason.
Thus, if the HT localization signal placed the enzyme in an earlier
temporal compartment than GT, it would have "first use" of the
N-acetyl lactosamine substrate. However, such a "first use" if it
occurred, was not sufficient to adequately reduce GT. Two chimeric
glycosyltransferases were constructed using PCR wherein the
cytoplasmic tails of GT and HT were switched. The two chimeras
constructed are shown in FIG. 1: ht-GT which consisted of the
NH.sub.2 terminal cytoplasmic tail of HT attached to the
transmembrane, stem and catalytic domains of GT; and gt-HT which
consisted of the NH.sub.2 terminal cytoplasmic tail of GT attached
to the transmembrane, stem and catalytic domains of HT. The
chimeric cDNAs were subcloned into the eukaryotic expression vector
pcDNAI and used in transfection experiments.
[0075] The chimeric cDNAs encoding ht-GT and gt-HT were initially
evaluated for their ability to induce glycosyltransferase
expression in COS calls, as measured by the surface expression of
the appropriate sugar using lectins. Forty-eight hours after
transfection COS calls were tested by immunofluorescence for their
expression of Gal-.alpha.(1,3)-Gal or H substance (Table 1 &
FIG. 2). The staining with IB4 (lectin specific for
Gal-.alpha.(1,3)-Gal) in cells expressing the chimera ht-GT (30% of
cells stained positive) was indistinguishable from that of the
normal GT staining (30%) (Table 1 & FIG. 2). Similarly the
intense cell surface fluorescence seen with UEAI staining (the
lectin specific for H substance) in cells expressing gt-HT (50%)
was similar to that seen in cells expressing wild-type pHT (50%)
(Table 1 & FIG. 2). Furthermore, similar levels of mRNA
expression of the glycosyltransferases GT and HT and chimeric
glycosyltransferases ht-GT and gt-HT were seen in Northern blots of
total RNA isolated from transfected cells (FIG. 3). Thus both
chimeric glycosyltransferases are efficiently expressed in COS
cells and are functional indeed there was no detectable difference
between the chimeric and normal glycosyltransferases.
[0076] Glycosyltransferase Activity in Cells Transfected with
Chimeric cDNAs Encoding ht-GT and gt-HT
[0077] To determine whether switching the cytoplasmic tails of GT
and HT altered the kinetics of enzyme function, we compared the
enzymatic activity of the chimeric glycosyltransferases with those
of the normal enzymes in COS cells after transfection of the
relevant cDNAs. By making extracts from transfected COS cells and
performing GT or HT enzyme assays we found that N-acetyl
lactosamine was galactosylated by both GT and the chimeric enzyme
ht-GT (FIG. 4. panel A) over a the 1-5 mM range of substrate
concentrations. Lineweaver-Burk plots showed that both GT and ht-GT
have a similar apparent Michealis-Menten constant of Km 2. 6 mM for
N-acetyl lactosamine (FIG. 4. panel B). Further HT, and the
chimeric enzyme gt-HT were both able to fucosylate
phenyl-B-D-galactoside over a range of concentrations (7.5-25 mM)
(FIG. 4 panel C) with a similar Km of 2.3 mM (FIG. 4 panel D), in
agreement with the reported Km of 2.4 mM for HT (25). Therefore the
chimeric glycosyltransferases ht-GT and gt-HT are able to utilize
N-acetyl lactosamine (ht-GT) and phenyl-B-D-galactoside (gt-HT) in
the same way as the normal glycosyltransferases, thus switching the
cytoplasmic domains of GT and HT does not alter the function of
these glycosyltransferases and if indeed the cytoplasmic tail is
the localization signal then both enzymes function as well with the
GT signal as with the HT signal.
[0078] Switching Cytoplasmic Domains of GT and HT Results in a
reversal of the "Dominance" of the Glycosyltransferases
[0079] The cDNAs encoding the chimeric transferases or normal
transferases were simultaneously co-transfected into COS cells and
after 48 h the cells were stained with either IB4 or UEA1 lectin to
detect Gal-.alpha.(1,3)-Gal and H substance respectively on the
cell surface (Table 1 & FIG. 5). COS cells co-transfected with
cDNAs for ht-GT+gt-HT (FIG. 5 panel C) showed 30% cells staining
positive with IB4 (Table 1) but no staining on cells co-transfected
with cDNAs for GT+HT (3%) (FIG. 5 panel A). Furthermore staining
for H substance on the surface of ht-GT+gt-HT co-transfectants gave
very few cells staining positive (5%) (FIG. 5 panel D) compared to
the staining seen in cells co-transfected with cDNAs for the normal
transferases GT+HT (50%) (FIG. 5 panel B), ie. the expression of
Gal-.alpha.(1,3)-Gal now dominates over that of H. Clearly,
switching the cytoplasmic tails of GT and HT led to a complete
reversal in the glycosylation pattern seen with the normal
transferases i.e. the cytoplasmic tail sequences dictate the
pattern of carbohydrate expression observed.
[0080] That exchanging the cytoplasmic tails of GT and HT reverses
the dominance of the carbohydrate epitopes points to the
glycosyltransferases being relocalized within the Golgi. To address
this question, experiments were performed with cDNAs encoding
glycosyltransferases with the same cytoplasmic tail: COS cells
transfected with cDNAs encoding HT+ht-GT stained strongly with both
UEA1 (50%) and 1B4 (30%) (Table 1 & FIG. 5 panels E, F), the
difference in staining reflecting differences in transfection
efficiency of the cDNAs. Similarly cells transfected with cDNAs
encoding GT+gt-HT also stained positive with UEAI (50%) and IB4
(30%) (Table 1 & FIG. 5 panel G, H). Thus, glycosyltransferases
with the same cytoplasmic tail leads to equal cell surface
expression of the carbohydrate epitopes, with no "dominance" of one
glycosyltransferase over the other observed, and presumably the
glycosyltransferases localised at the same site appear to compete
equally for the substrate.
[0081] In COS cells the levels of transcription of the cDNAs of
chimeric and normal glycosyltransferases were essentially the same
(FIG. 3) and the immunofluorescence pattern of COS cells expressing
the chimeric glycosyltransferases ht-GT and gt-HT showed the
typical staining pattern of the cell surface Gal-.alpha.(1,3)-Gal
and H substance respectively (Table 1 & FIG. 2), the pattern
being indistinguishable from that of COS cells expressing normal GT
and HT. Our studies showed that the Km of ht-GT for N-acetyl
lactosamine was identical to the Km of GT for this substrate,
similarly the Km of gt-HT for phenylBDgalactoside was approximately
the same as the Km of HT for phenylbDgalactoside (FIG. 3). These
findings indicate that the chimeric enzymes are functioning in a
cytoplasmic tail--independent manner, such that the catalytic
domains are entirely functional, and are in agreement with those of
Henion et al (23), who showed that an NH2 terminal truncated
marmoset GT (including truncation of the cytoplasmic and
transmembrane domains) maintained catalytic activity and confirmed
that GT activity is indeed independent of the cytoplasmic domain
sequence.
[0082] If the Golgi localization signal for GT and HT is contained
entirely within the cytoplasmic domains of the enzymes, then
switching the cytoplasmic tails between the two transferases should
allow a reversal of the order of glycosylation. Co-transfection of
COS cells with cDNA encoding the chimeric glycosyltransferases
ht-GT and gt-HT caused a reversal of staining observed with the
wild type glycosyltransferases (FIG. 5), demonstrating that the
order of glycosylation has been altered by exchanging the
cytoplasmic tails. Furthermore, co-transfection with cDNA encoding
glycosyltransferases with the same cytoplasmic tails (i. e.
HT+ht-GT and GT+gt-HT) gave rise to equal expression of both
Gal-.alpha.(1,3)-Gal and H substance (FIG. 5). The results imply
that the cytoplasmic tails of GT and HT are sufficient for the
localization and retention of these two enzymes within the
Golgi.
[0083] To date only twenty or so of at least one hundred predicted
glycosyltransferases have been cloned and few of these have been
studied with respect to their Golgi localization and retention
signals (34). Studies using the elongation transferase
N-acetylglucosaminyltransferase I (33-37), the terminal
transferases a(2,6)sialyltransferase (24-26) and
.beta.(1,4)galactosyltransferase (38-40) point to residues
contained within the cytoplasmic tail, transmembrane and flanking
stem regions as being critical for Golgi localization and
retention. There are several examples of localization signals
existing within cytoplasmic tail domains of proteins including the
KDEL and KKXX motifs in proteins resident within the endoplasmic
reticulum, (41,42) the latter motif also having been identified in
the cis Golgi resident protein ERGIC-53 (43) and a di-leucine
containing peptide motif in the mannose-6-phosphate receptor which
directs the receptor from the trans-Golgi network to endosomes
(44). These motifs are not present within the cytoplasmic tail
sequences of HT or GT or in any other reported glycosyltransferase.
To date a localization signal in Golgi resident
glycosyltransferases has not been identified and while there is
consensus that transmembrane domains are important in Golgi
localization, it is apparent that this domain is not essential for
the localization of all glycosyltransferases, as shown by the study
of Munro (45) where replacement of the transmembrane domain of
.alpha.(2,6)sialyltransferase in a hybrid protein with a
poly-leucine tract resulted in normal Golgi retention.
[0084] Dahdal and Colley (46) also showed that sequences in the
transmembrane domain were not essential to Golgi retention. This
study is the first to identify sequence requirements for the
localization of .alpha.(1,2)fucosyltransferase and
.alpha.(1,3)galactosyltransferase within the Golgi. It is
anticipated that other glycosyltransferases will have similar
localization mechanisms.
EXAMPLE 2
Use of Secretor in Construction of a Chimeric Enzyme
[0085] A construct is made using PCR and subcloning as described in
Example 1, such that amino acids #1 to #6 of the pig
.alpha.(1,3)-galactosyltransferase (MNVKGR) (SEQ ID No. 14) replace
amino acids #1 to 5 of the pig secretor (FIG. 6). Constructs are
tested as described in Example 1.
EXAMPLE 3
Use of Pig H Transferase in Construction of a Chimeric Enzyme
[0086] A construct is made using PCR and subcloning as described in
Example 1, such that amino acids #1 to #6 of the pig
.alpha.(1,3)-galactosyltransferase (MNVKGR) (SEQ ID No. 14) replace
amino acids #1 to 8 of the pig H transferase (FIG. 7). Constructs
are tested as described in Example 1.
EXAMPLE 4
Generation of Pig Endothelial Cells Expressing Chimeric
.alpha.(1,2)fucosyltransferase
[0087] The pig endothelial cell line PIEC expressing the chimeric
.alpha.l,2fucosyltransferase was produced by lipofectamine
transfection of pgtHT plasmid DNA (20 .mu.g) and pSV2NEO (2 .mu.g)
and selecting for stable integration by growing the transfected
PIEC in media containing G418 (500 .mu.g/ml; Gibco-BRL,
Gaithersburg, Md.). Fourteen independent clones were examined for
cell surface expression of H substance by staining with UEA-1
lectin. >95% of cells of each of these clones were found to be
positive. FIG. 8 shows a typical FACS profile obtained for these
clones.
EXAMPLE 5
Production of Transgenic Mice Expressing Chimeric (x(1,2)
fucosyltransferase
[0088] A NruI/NotI DNA fragment, encoding the full length chimeric
.alpha.l,2fucosyltransferase, was generated utilizing the
Polymerase Chain Reaction and the phHT plasmid using the
primers:
[0089] 5' primer homologous to the 5'UTR: TABLE-US-00001
5'-TTCGCGAATGAATGTCAAAGGAAGACTCTG, (SEQ ID No. 9)
in which the double-underlined sequence contains a unique NruI
site;
[0090] 3' primer homologous to the 3'UTR:
[0091] 5'-GGCGGCCGCTCAGATGTTATTTCTAACCAAAT
[0092] (SEQ ID No. 10) the double-underlined sequence contains a
Notl site
[0093] The DNA was purified on gels, electroeluted and subcloned
into a NruI/NotI cut genomic H-2Kb containing vector resulting in
the plasmid clone (pH-2Kb-gtHT) encoding the chimeric
.alpha.(1,2)-fucosyltransferase gene directionally cloned into exon
1 of the murine H-2Kb gene, resulting in a transcript that
commences at the H-2Kb transcriptional start site, continuing
through the gtHT cDNA insert. The construct was engineered such
that translation would begin at the initiation codon (ATG) of the
hHT cDNA and terminate at the in-phase stop codon (TGA).
[0094] DNA was prepared for microinjection by digesting pH-2Kb-hHT
with XhoI and purification of the H-2Kb-hHT DNA from vector by
electrophoretic separation in agarose gels, followed by extraction
with chloroform, and precipitation in ethanol to decontaminate the
DNA. Injections were performed into the pronuclear membrane of
(C57BL/6xSJL)Fl zygotes at concentrations between 2-5 ng/ml, and
the zygotes transferred to pseudopregnant (C57BL/6xSJL)Fl
females.
[0095] The presence of the transgene in the live offspring was
detected by dot blotting. 5 mg of genomic DNA was transferred to
nylon filters and hybridized with the insert from gtHT, using a
final wash at 68.degree. C. in 0.1.times.SSC/1% SDS. FIG. 9 shows
the results of testing 12 live offspring, with two mice having the
transgenic construct integrated into the genome. Expression of
transgenic protein is examined by estimating the amount of UEAI
lectin (specific for H substance) or anti-H mAb required to
haemagglutinate red blood cells from transgenic mice.
Hemagglutination in this assay demonstrates transgene
expression.
[0096] It will be apparent to the person skilled in the art that
while the invention has been described in some detail for the
purposes of clarity and understanding, various modifications and
alterations to the embodiments and methods described herein may be
made without departing from the scope of the inventive concept
disclosed in this specification.
[0097] References cited herein are listed on the following pages,
and are incorporated herein by this reference. TABLE-US-00002 TABLE
1 EXPRESSION OF GAL-.alpha.(1,3)GAL AND H SUBSTANCE BY COS CELLS
TRANSFECTED WITH cDNAs ENCODING NORMAL AND CHIMERIC
GLYCOSYLTRANSFERASES COS calls transfected % IB4 positive % UEAI
positive with cDNA encoding: calls cells GT 30 0 HT 0 50 ht - GT 30
0 gt - HT 3 50 GT + HT 3 50 ht - GT + gt - HT 33 5 GT + gt - HT 30
30 GT + ht - GT 30 0 HT + ht - GT 30 30 HT + gt - HT 0 50 Mock 0
0
[0098] Transfected COS cells were stained with FITC-labelled IB4
(lectin specific for Gal-.alpha.(1,3)Gal or UEAI (lectin specific
for H substance) and positive staining cells were visualized and
counted by fluorescence microscopy. Results are from at least three
replicates.
REFERENCES
[0099] 1. Leventhal, J R et al. Complement depletion prolongs
discordant cardiac xenograft survival in rodents nad non-human
primates. Transplantn Prod. 25, 398-399 (1993).
[0100] 2. Pruitt, S et al. The effect of soluble complement
receptor type 1 on hyperacute rejection of porcine xenografts.
Transplantation 57, 363-370 (1994).
[0101] 3. Leventhal, J R et al. Removal of baboon and human
antiporcine IgG and IgM natural antibodies by immunoabsorption.
Transplantation 59, 294-300 (1995).
[0102] 4. Brewer, R J et al. Depletion of preformed natural
antibody in primates for discordant xenotransplantation by
continuous donor organ plasma perfusion. Transplantation Proac. 25,
385-386 (1993).
[0103] 5. McCurry, K R et al. Human complement regulatory proteins
protect swine-to-primate cardiac xenografts from humoral injury.
Nature Med. 1, 423-427 (1995).
[0104] 6. Fodor, W L et al. Expression of a functional human
complement inhibitor in a transgenic pig as a model for the
prevention of xenogeneic hyperacute organ rejection. Proc. Natn.
Acad. Sci USA 91, 11153-11157 (1994).
[0105] 7. Rosengard, A M et al. Tissue expression of the human
complement inhibitor decay accelerating factor in transgenic pigs.
Transplantation 59, 1325-1333 (1995).
[0106] 8. Sandrin, M S, Vaughan, H A, Dabkowski, P L &
McKenzie, I F C. Anti-pig IgM antibodies in human serum reacts
predominantly with Gal(al,3)Gal epitopes. Prod. Natn. Acad. Sci USA
90, 11391-11395 (1993).
[0107] 9. Sandrin, M S, Vaughan, H A & McKenzie, I F C.
Identification of Gal(al, 3)Gal as the major epitope of
pig-to-human vascularised xenografts. Transplantation Rev. 8,
134-149 (1994).
[0108] 10. Sandrin, M S & McKenzie, I F C. Gal(al,3)Gal, the
major xenoantigen(s) recognised in pigs by human natural
antibodies. Immunol. Rev. 141. 169-190 (1994).
[0109] 11. Cooper, D K C et al. Identification of agalactosyl and
other carbohydrate epitopes that are bound by human anti-pig
antibodies. Relevance to discordant xenografting in man.
Transplantation Immun. 1. 198-205 (1993).
[0110] 12. Cooper, D K C, Koren, E & Oriol, R. Oligosaccharides
and discordant xenotransplantation. Immunol. Rev. 141. 31-58
(1994).
[0111] 13. Good, A H et al. Identification of carbohydrate
structures that bind antiporcine antibodies: Implications for
discordant xenografting in humans. Transplantation Proc. 24.
559-562 (1992).
[0112] 14. Galili, U., Clark, M R., Shohet, S B., Buehler, J &
Macher, B A. Evolutionary relationship between the natural anti-Gal
antibody and the Galal-3Gal epitope inprimates. Proc. Natn. Acad.
Sci USA 84. 1369-1373 (1987).
[0113] 15. Galili, U., Shohet, S B., Korbin, E., Stults, C L M
& Macher, B A. Man, apes and Old world monkeys differ from
other mammals in the expression of the a-galactosyl epitopes on
nucleated cells. J. biol. Chem. 263. 17755-17762 (1988).
[0114] 16. Larsen, R D et al. Isolation of a cDNA encoding a murine
UDPgalactose:b-D-galctosyl-1,4-N-acetyl-glucosaminide-1,3-galactosyltrans-
ferase: Expression cloning by gene transfer. Proc. natn. Acd. Sci.
USA 86. 8227-8231d (1989).
[0115] 17. Joziasse, D H., Shaper, J H., Kim D., Van den Eijnden, D
H & Shaper, J H. Murine al,3 galactosyltransferase a single
gene locus specifies four isoforms of the enzyme by alternative
splicing. J. biol. Chem. 267, 5534-5541 (1992).
[0116] 18. Joziasse, D H, Shaper, J H, Van den Eijnden, D H, Van
Tunen, A J & Shaper, N L. bovine al,3 galactosyltransferase:
Isolation and characterization of a cDNA cone. Identification of
homologous sequences in human genomic DNA. J. biol, Chem. 264.
14290-14297 (1989).
[0117] 19. Sandrin, M S, Dabkowski, P I, Henning, M M, Mouhtouris,
E & McKenzie, I F C. Characterization of cDNA clones for
porcine al,3 galactosyltransferase. The enzyme generating the
Gal(al,3)Gal epitope. Xenotransplantation 1, 81-88 (1994).
[0118] 20. Joziasse, D H, Shaper, J H, Jabs, F W & Shaper, N L.
Characterization of an al,3-galactosyltransferase homologue on
human chromosome 12 that is organized as a processed pseudogene. J.
biol. Chem. 266. 6991-6998 (1991).
[0119] 21. Larsen, R D, Riverra-Marrero, C A, Ernst, L K, Cummings,
R D & Lowe, J B. Frameshift and non sense mutations in a human
genomic sequence homologous toa murine UDP-Gal:b-D-Gal
1,4-D-GlcNAcal,3-galactosyl-transferase cDNA. J. biol. Chem. 265.
7055-7061 (1990).
[0120] 22. Kiote, C et al. Introduction of a(1,2)fucosyltransferase
and its effect on a-Gal epitopes in transgenic pig.
Xenotransplantation 3:81-86.
[0121] 23. Sandrin, M. S., Dabkowski, P. L., Henning, M. M.,
Mouhtouris, E., and McKenzie, 1. F. C. (1994) Xenotransplantation
1, 81-88
[0122] 24. Cohney, S., Mouhtouris, E., McKenzie, I. F. C., and
Sandrin, M. S. (1996) Immunogenetics 44(1), 76-79
[0123] 25. Larsen, R. D., Ernst, L. K., Nair, R. P., and Lowe, J.
B. (1990) Proc. Natl. Acad. Sci. USA 87, 6674-6678
[0124] 26. Sandrin, M. S., Vaughan, H. A., Dabkowski, P. L., and
McKenzie, I. F. C. (1993) PrOC. Natl. Acad. Sci. USA 90,
11391-11395
[0125] 27. Hayes, C. E., and Goldstein, I. J. (1974) J. Biol. Chem.
6, 1904-1914
[0126] 28. Matsumoto, I., and Osowa, T. (1969) Biochim. Biophys.
Acta 194, 180-189
[0127] 29. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254
[0128] 30. Rajan, V. R., Larsen, R. D., Ajmera, S., Ernst, L. K.,
and Lowe, J. B. (1989) J. Biol. Chem 264, 11158-11167
[0129] 31. van der Eijnden, D. H., Blanken, W. M., Winterwerp, H.,
and Schiphorst, W. E. C. M. (1983) Eur. J. Biochem. 134,
523-530
[0130] 32. Sandrin, M. S., Fodor, W. F., Mouhtouris,. E., Osman,
N., Cohney, S. C., Rollins,S. A., Guilmette, E. R., Setter, E.,
Squinto, S. P., and McKenzie, I. F. C. (1995) Nature Med. 1,
1261-1267
[0131] 33. Henion, T. R., Macher, B. A., Anaraki, F., and Galili,
U. (1994) Glycobiology 4, 193-201
[0132] 34. Schachter, H. (1994) in Molecular Glycobiology (Fukuda,
M., and Hindsgaul, 0., eds), pp. 88-162, Oxford University Press,
Oxford
[0133] 35. Burke, J., Pettitt, J. M., Schachter, H., Sarkar, M.,
and Gleeson, P. A. (1992) J. Biol. Chem. 267,-24433-24440
[0134] 36. Tang, B. L., Wong, S. H., Low, S. H., and Hong, W.
(1992) J. Biol. Chem. 267, 10122
[0135] 37. Nilsson, T., Pypeart, M., Hoe, M. H., Slusarewicz, P.,
Berger, E., and Warren, G. (1993) J. Cell Biol. 120, 5
[0136] 38. Nilsson, T., Lucocq, J. M., Mackay, D., and Warren, G.
(1991) EMBO J. 10, 3567-3575
[0137] 39. Aoki, D., Lee, N., Yamaguchi, N., Dubois, C., and
Fukuda, M. N. (1992) Proc. natl. Acad. Sci. USA 89, 4319-4323
[0138] 40. Teasdale, R. D., D'Agostaro, G. D., and Gleeson, P. A.
(1992) J. Biol. Chem. 267, 4084-4096
[0139] 41. Pelham, H. R. (1990) Trends Biochem. Sci. 15,
483-486
[0140] 42. Jackson, M. R., Nilsson, T., and Peterson, P. A. (1990)
EMBO J. 9, 3153-3162
[0141] 43. Kappeler, F., Itin, C., Schindler, R., and Hauri, H.-P.
(1994) J. Biol. Chem. 269, 6279-6281
[0142] 44. Johnson, K. F., and Kornfeld, S. (1992) J. Biol. Chem.
267, 17110-17115
[0143] 45. Munro, S. (1991) EMBO J. 10, 3577-3588
[0144] 46. Dahdal, R. Y., and Colley, K. J. (1993) J. Biol. Chem.
268, 26310-26319
Sequence CWU 1
1
16 1 1043 DNA Sus Domesticus 1 ctacagccat gctcagcatg caggcatcct
tcttcttccc cacgggtccc ttcatcctct 60 ttgtcttcac ggcttccacc
atatttcacc ttcagcagag gatggtgaag attcaaccca 120 cgtgggagtt
acagatggtg acgcaggtga ccacagagag cccctcgagc ccccagctga 180
agggcatgtg gacgatcaat gccatcggcc gcctggggaa ccagatgggg gagtacgcca
240 ccctgtacgc gctggccagg atgaacgggc ggccggcctt catcccgccc
gagatgcaca 300 gcacgctggc ccccatcttc aggatcaccc tcccggtcct
gcacgccagc acggcccgca 360 ggatcccctg gcagaactac cacctgaacg
actggatgga ggagcggtac cgccacatcc 420 cgggggagta cgtgcgcctc
acgggctacc cctgctcctg gaccttctac caccacctgc 480 gcaccgagat
cctccgggag ttcaccctgc ataaccacgt gcgcgaggag gcccaggatt 540
tcctgcgggg tctgcgggtg aacgggagcc gaccgagtac ctacgtgggg gtgcacgtgc
600 gccgggggga ctacgtgcac gtgatgccca acgtgtggaa gggcgtggtg
gccgaccggc 660 ggtacctgga gcaggccctg gactggttcc gggctcgcta
ccgctccccc gtctttgtgg 720 tctccagcaa cggcatggcc tggtgtcggg
aaaacatcaa tgcctcgcgc ggcgatgtgg 780 tgtttgccgg caatggcatc
gagggctccc ccgccaaaga cttcgcgctg ctcacgcagt 840 gtaaccacac
tgtcatgacc attggcacgt tcgggatctg ggccgcctac cttgctggtg 900
gagagaccat ctacctggcc aattacacgc tcccggactc tcccttcctc aaactcttta
960 agcccgaggc agccttcctg cccgagtgga ttgggatcga ggcagacctg
tccccactcc 1020 ttaagcactg atgtcggctg tcc 1043 2 340 PRT Sus
Domesticus 2 Met Leu Ser Met Gln Ala Ser Phe Phe Phe Pro Thr Gly
Pro Phe Ile 1 5 10 15 Leu Phe Val Phe Thr Ala Ser Thr Ile Phe His
Leu Gln Gln Arg Met 20 25 30 Val Lys Ile Gln Pro Thr Trp Glu Leu
Gln Met Val Thr Gln Val Thr 35 40 45 Thr Glu Ser Pro Ser Ser Pro
Gln Leu Lys Gly Met Trp Thr Ile Asn 50 55 60 Ala Ile Gly Arg Leu
Gly Asn Gln Met Gly Glu Tyr Ala Thr Leu Tyr 65 70 75 80 Ala Leu Ala
Arg Met Asn Gly Arg Pro Ala Phe Ile Pro Pro Glu Met 85 90 95 His
Ser Thr Leu Ala Pro Ile Phe Arg Ile Thr Leu Pro Val Leu His 100 105
110 Ala Ser Thr Ala Arg Arg Ile Pro Trp Gln Asn Tyr His Leu Asn Asp
115 120 125 Trp Met Glu Glu Arg Tyr Arg His Ile Pro Gly Glu Tyr Val
Arg Leu 130 135 140 Thr Gly Tyr Pro Cys Ser Trp Thr Phe Tyr His His
Leu Arg Thr Glu 145 150 155 160 Ile Leu Arg Glu Phe Thr Leu His Asn
His Val Arg Glu Glu Ala Gln 165 170 175 Asp Phe Leu Arg Gly Leu Arg
Val Asn Gly Ser Arg Pro Ser Thr Tyr 180 185 190 Val Gly Val His Val
Arg Arg Gly Asp Tyr Val His Val Met Pro Asn 195 200 205 Val Trp Lys
Gly Val Val Ala Asp Arg Arg Tyr Leu Glu Gln Ala Leu 210 215 220 Asp
Trp Phe Arg Ala Arg Tyr Arg Ser Pro Val Phe Val Val Ser Ser 225 230
235 240 Asn Gly Met Ala Trp Cys Arg Glu Asn Ile Asn Ala Ser Arg Gly
Asp 245 250 255 Val Val Phe Ala Gly Asn Gly Ile Glu Gly Ser Pro Ala
Lys Asp Phe 260 265 270 Ala Leu Leu Thr Gln Cys Asn His Thr Val Met
Thr Ile Gly Thr Phe 275 280 285 Gly Ile Trp Ala Ala Tyr Leu Ala Gly
Gly Glu Thr Ile Tyr Leu Ala 290 295 300 Asn Tyr Thr Leu Pro Asp Ser
Pro Phe Leu Lys Leu Phe Lys Pro Glu 305 310 315 320 Ala Ala Phe Leu
Pro Glu Trp Ile Gly Ile Glu Ala Asp Leu Ser Pro 325 330 335 Leu Leu
Lys His 340 3 1098 DNA Sus Domesticus 3 atgtgggtcc ccagccgccg
ccacctctgt ctgaccttcc tgctagtctg tgttttagca 60 gcaattttct
tcctgaacgt ctatcaagac ctcttttaca gtggcttaga cctgctggcc 120
ctgtgtccag accataacgt ggtatcatct cccgtggcca tattctgcct ggcgggcacg
180 ccggtacacc ccaacgcctc cgattcctgt cccaagcatc ctgcctcctt
ttccgggacc 240 tggactattt acccggatgg ccggtttggg aaccagatgg
gacagtatgc cacgctgctg 300 gccctggcgc agctcaacgg ccgccaggcc
ttcatccagc ctgccatgca cgccgtcctg 360 gcccccgtgt tccgcatcac
gctgcctgtc ctggcgcccg aggtagacag gcacgctcct 420 tggcgggagc
tggagcttca cgactggatg tccgaggatt atgcccactt aaaggagccc 480
tggctgaagc tcaccggctt cccctgctcc tggaccttct tccaccacct ccgggagcag
540 atccgcagcg agttcaccct gcacgaccac cttcggcaag aggcccaggg
ggtactgagt 600 cagttccgtc taccccgcac aggggaccgc cccagcacct
tcgtgggggt ccacgtgcgc 660 cgcggggact atctccgtgt gatgcccaag
cgctggaagg gggtggtggg tgacggcgct 720 tacctccagc aggctatgga
ctggttccgg gcccgatacg aagcccccgt ctttgtggtc 780 accagcaacg
gcatggagtg gtgccggaag aacatcgaca cctcccgggg ggacgtgatc 840
tttgctggcg atgggcggga ggccgcgccc gccagggact ttgcgctgct ggtgcagtgc
900 aaccacacca tcatgaccat tggcaccttc ggcttctggg ccgcctacct
ggctggtgga 960 gataccatct acttggctaa cttcaccctg cccacttcca
gcttcctgaa gatctttaaa 1020 cccgaggctg ccttcctgcc cgagtgggtg
ggcattaatg cagacttgtc tccactccag 1080 atgttggctg ggccttga 1098 4
365 PRT Sus Domesticus 4 Met Trp Val Pro Ser Arg Arg His Leu Cys
Leu Thr Phe Leu Leu Val 1 5 10 15 Cys Val Leu Ala Ala Ile Phe Phe
Leu Asn Val Tyr Gln Asp Leu Phe 20 25 30 Tyr Ser Gly Leu Asp Leu
Leu Ala Leu Cys Pro Asp His Asn Val Val 35 40 45 Ser Ser Pro Val
Ala Ile Phe Cys Leu Ala Gly Thr Pro Val His Pro 50 55 60 Asn Ala
Ser Asp Ser Cys Pro Lys His Pro Ala Ser Phe Ser Gly Thr 65 70 75 80
Trp Thr Ile Tyr Pro Asp Gly Arg Phe Gly Asn Gln Met Gly Gln Tyr 85
90 95 Ala Thr Leu Leu Ala Leu Ala Gln Leu Asn Gly Arg Gln Ala Phe
Ile 100 105 110 Gln Pro Ala Met His Ala Val Leu Ala Pro Val Phe Arg
Ile Thr Leu 115 120 125 Pro Val Leu Ala Pro Glu Val Asp Arg Glu Ala
Ile Trp Arg Glu Leu 130 135 140 Glu Leu His Asp Trp Met Ser Glu Asp
Tyr Ala His Leu Lys Arg Pro 145 150 155 160 Trp Leu Lys Leu Thr Gly
Phe Pro Cys Ser Trp Thr Phe Phe His His 165 170 175 Leu Arg Glu Gln
Ile Arg Ser Glu Phe Thr Leu His Asp His Leu Arg 180 185 190 Gln Glu
Ala Gln Gly Val Leu Ser Gln Phe Arg Leu Pro Arg Thr Gly 195 200 205
Asp Arg Pro Ser Thr Phe Val Gly Val His Val Arg Arg Gly Asp Tyr 210
215 220 Leu Arg Val Met Pro Lys Arg Trp Lys Gly Val Val Gly Asp Gly
Ala 225 230 235 240 Tyr Leu Gln Gln Ala Met Asp Trp Phe Arg Ala Arg
Tyr Glu Ala Pro 245 250 255 Val Phe Val Val Thr Ser Asn Gly Met Glu
Trp Cys Arg Lys Asn Ile 260 265 270 Asp Thr Ser Arg Gly Asp Val Ile
Phe Ala Gly Asp Gly Arg Glu Ala 275 280 285 Ala Pro Ala Arg Asp Phe
Ala Leu Leu Val Gln Cys Asn His Thr Ile 290 295 300 Met Thr Ile Gly
Thr Phe Gly Phe Trp Ala Ala Tyr Leu Ala Gly Gly 305 310 315 320 Asp
Thr Ile Tyr Leu Ala Asn Phe Thr Leu Pro Thr Ser Ser Phe Leu 325 330
335 Lys Ile Pro Lys Pro Glu Ala Ala Phe Leu Phe Glu Trp Val Gly Ile
340 345 350 Asn Ala Asp Leu Ser Pro Leu Gln Met Leu Ala Gly Pro 355
360 365 5 54 DNA Artificial Sequence Chimeric, Homo Sapiens and Sus
Domesticus 5 gcggatccat gtggctccgg agccatcgtc aggtggttct gtcaatgctg
cttg 54 6 39 DNA Artificial Sequence Chimeric, Homo Sapiens and Sus
Domesticus 6 gctctagagc gtcagatgtt atttctaacc aaattatac 39 7 45 DNA
Artificial Sequence Chimeric, Homo Sapiens and Sus Domesticus 7
gcggatccat gaatgtcaaa ggaagactct gcctggcctt cctgc 45 8 35 DNA
Artificial Sequence Chimeric, Homo Sapiens and Sus Domesticus 8
gctctagagc ctcaaggctt agccaatgtc cagag 35 9 30 DNA Artificial
Sequence Chimeric, Homo Sapiens and Sus Domesticus 9 ttcgcgaatg
aatgtcaaag gaagactctg 30 10 32 DNA Artificial Sequence Chimeric,
Homo Sapiens and Sus Domesticus 10 ggcggccgct cagatgttat ttctaaccaa
at 32 11 6 PRT Sus scrofa 11 Met Asn Val Lys Gly Arg 1 5 12 6 PRT
Mus musculus 12 Met Asn Val Lys Gly Lys 1 5 13 6 PRT Unknown Bovine
13 Met Val Val Lys Gly Lys 1 5 14 6 PRT Sus scrofa 14 Met Asn Val
Lys Gly Arg 1 5 15 4 PRT Unknown Bovine 15 Lys Asp Glu Leu 1 16 4
PRT Unknown Bovine misc_feature (3)..(4) Xaa can be any naturally
occurring amino acid 16 Lys Lys Xaa Xaa 1
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