U.S. patent application number 12/120623 was filed with the patent office on 2008-09-18 for compositions and methods for regulating receptor clustering.
This patent application is currently assigned to MOUNT SINAI HOSPITAL. Invention is credited to Michael Demetriou, James Dennis.
Application Number | 20080227750 12/120623 |
Document ID | / |
Family ID | 22993657 |
Filed Date | 2008-09-18 |
United States Patent
Application |
20080227750 |
Kind Code |
A1 |
Dennis; James ; et
al. |
September 18, 2008 |
COMPOSITIONS AND METHODS FOR REGULATING RECEPTOR CLUSTERING
Abstract
The invention relates to isolated complexes comprising one or
more galectin associated with a Mgat5 modified glycan or
polylactosamine modified glycan, and isolated lectin-Mgt5 modified
glycan lattice comprising an array of mulitvalent interactions
among lectins, Mgat5 modified glycans, polylactosamine modified
glycans, and/or glycoproteins. Methods for evaluating a test
compound for its ability to regulate receptor clustering through
glycans on cell surfaces; and methods for regulating receptor
clustering on cell surfaces comprising altering glycans on the cell
surface associated with receptor clustering are also disclosed.
Inventors: |
Dennis; James; (Etobicoke,
CA) ; Demetriou; Michael; (Irvine, CA) |
Correspondence
Address: |
HOWSON AND HOWSON
SUITE 210, 501 OFFICE CENTER DRIVE
FT WASHINGTON
PA
19034
US
|
Assignee: |
MOUNT SINAI HOSPITAL
Toronto
CA
|
Family ID: |
22993657 |
Appl. No.: |
12/120623 |
Filed: |
May 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10250935 |
Dec 15, 2003 |
7396529 |
|
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PCT/CA02/00002 |
Jan 11, 2002 |
|
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12120623 |
|
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60261516 |
Jan 12, 2001 |
|
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Current U.S.
Class: |
514/54 ; 435/29;
436/94; 536/123.1 |
Current CPC
Class: |
C07K 14/4726 20130101;
Y10T 436/143333 20150115; G01N 33/5008 20130101; G01N 2333/4724
20130101; G01N 2500/00 20130101; A61P 43/00 20180101; G01N 33/5091
20130101 |
Class at
Publication: |
514/54 ;
536/123.1; 436/94; 435/29 |
International
Class: |
A61K 31/715 20060101
A61K031/715; C07H 1/00 20060101 C07H001/00; C12Q 1/02 20060101
C12Q001/02; A61P 43/00 20060101 A61P043/00; G01N 33/00 20060101
G01N033/00 |
Claims
1. An isolated lectin-Mgat5 modified glycan lattice comprising an
array of multivalent interactions among lectins, Mgat5 modified
glycans, polylactosamine modified glycans, and/or glycoproteins
that are associated with receptor clustering.
2. An isolated lectin-Mgat5 modified glycan lattice as claimed in
claim 1 wherein the Mgt5 modified glycans and polylactosamine
modified glycans are part of glycoproteins of receptors.
3. A method for evaluating a compound for its ability to regulate
receptor clustering through a lectin-Mgat5 modified glycan lattice,
the method comprising (a) mixing a lectin-Mgat5 modified glycan
lattice as claimed in claim 1, or a lectin and one or more of a
Mgat5 modified glycan and polylactosamine modified glycan, and a
test compound, under conditions which maintain the lattice or
permit the formation of complexes between the lectin and one or
more of the Mgat5 modified glycan and polylactosamine modified
glycan; and (b) removing and/or detecting lectin-Mgat5 modified
glycan lattice, complexes, lectin, Mgat5 modified glycan, or
polylactosamine modified glycan.
4. A method as claimed in claim 3, wherein the lectin-Mgat5
modified glycan lattice is a galectin-Mgat5 modified glycan lattice
and the lectin is a galectin.
5. A method for (i) regulating receptor clustering on cell surfaces
or (ii) treating or preventing a condition associated with
decreased or increased receptor clustering or a receptor clustering
defect in a subject, the method comprising altering glycans
associated with receptor clustering.
6. A method as claimed in claim 5, wherein the receptor is selected
from the group consisting of a receptor that stimulates immune
reactions T cell receptor, Ig receptor, B cell receptor, NK
receptor, a member of the HER family of transmembrane receptor
tyrosine kinases, a cadherin receptor, an interleukin (IL)
receptor, TNF.gamma. receptor, and an integrin.
7. A method as claimed in claim 5. wherein the glycans are altered
by modulating one or more glycosyltransferase enzyme involved in
the synthesis of the glycans
8. A method as claimed in claim 7, wherein the glycans are Mgat5
modified glycans and the glycosyltransferase is Mgat5.
9. A method as claimed in claim 7, wherein the glycans are
polylactosamine modified glycans and the glycosyltransferase is
Mgat5.
10. A method for regulating receptor clustering on cell surfaces
comprising modulating Mgat5 activity, the amount of Mgat5 modified
glycans, polylactosamine modified glycans, or THE lectin-Mgat5
modified glycan lattice as claimed in claim 1, or the amount of
binding or interaction of one or more components of the
lectin-Mgat5 modified glycan lattice.
11. A method as claimed in claim 5, the method comprising
increasing the amount of Mgat5 modified glycans, polylactosamine
modified glycans, or lectin-Mgat5 modified glycan lattice on the
cell surface.
12. A method for restricting T cell receptor recruitment in
response to an agonist or increasing T cell activation threshold
comprising increasing Mgat5 modified glycans, polylactosamine
modified glycans, or the galectin-Mgat5 modified glycan lattice as
claimed in claim 1 on the surface of the cells, or enhancing the
interaction between one or more components of the galectin-Mgat5
modified glycan lattice.
13. A method as claimed in claim 12, wherein the glycans or lattice
are increased by increasing the amount or activity of one or more
glycosyltransferase enzyme.
14. A method as claimed in claim 5 which is for increasing receptor
clustering on a cell surface by decreasing the amount of Mgat5
modified glycans, polylactosamine modified glycans, or lectin-Mgat5
modified glycan lattice on the cell surface.
15. A method for lowering T cell activation threshold to agonists
or enhancing T cell receptor clustering comprising decreasing Mgat5
modified glycans, polylactosamine modified glycans, or
galectin-Mgat5 modified glycan lattice on the surface of the T
cells, or dissociating galectin from such glyeans or lattice
thereby lowering the T cell activation threshold.
16. A method as claimed in claim 15, wherein the glycans or lattice
are decreased by decreasing the amount of or activity of a
glycosyltransferase.
17. A method for treating or preventing a condition associated with
decreased or increased receptor clustering, or a receptor
clustering defect, comprising modulating Mgat5 activity, the amount
of Mgat5 modified glycans, polylactosamine modified glycans, or
lectin-Mgat5 modified glycan lattice, or the amount of binding or
interaction of one or more components of the lectin-MgatV modified
glycan lattice as claimed in claim 1.
18. A method for treating or preventing an autoimmune disease in a
subject comprising reducing T cell receptor clustering in the
subject by increasing the amount of Mgat5 modified glycans,
polylactosamine modified glycans, and/or the lectin-Mgat5 modified
glycan lattice as claimed in claim 1 on the surface of T cells of
the subject.
19. A method of diagnosing and monitoring conditions characterized
by an abnormality in receptor clustering comprising assaying for
Mgat5 modified glycans, the polylactosamine modified glycans,
lectin-Mgat5 modified glycan lattice as claimed in claim 1, or
alterations in such glycans or lattice, compared to a control.
20. A method of diagnosing and monitoring conditions characterized
by an abnormality or defect of receptor clustering involving the
interaction of a galectin and Mgat5 modified glycan or
polylactosamine modified glycan comprising determining the presence
of one or more complex as claimed in claim 1, a Mgat5 modified
glycan, a polylactosamine modified glycan, or one or more of an
altered Mgat5 modified glycan, polylactosamine modified glycan, or
a galectin-Mgat5 modified glycan lattice.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/250,935, filed Dec. 15, 2003, which is a US national
stage of International Patent Application No. PCT/CA02/00002, filed
Jan. 11, 2002, which claims the benefit of the priority of U.S.
Provisional Patent Application No. 60/261,516, filed Jan. 12, 2001,
now abandoned.
FIELD OF THE INVENTION
[0002] The invention relates to complexes, lattices, compositions
and methods for regulating receptor clustering on cell
surfaces.
BACKGROUND OF THE INVENTION
[0003] N- and O-linked glycans are found on both cell-surface and
secreted proteins, many of which control proliferation and cell
fate decisions in animals. Tissue-specific expression of
glycosyltransferases is a significant factor controlling the glycan
profiles observed in differentiated cells (Paulson, Jc and Colley K
J, J Biol Chem 1989, 264:17615-17618). In addition, many
glycosyltransferases compete for acceptor intermediates causing
bifurcations of the pathways and additional structural complexity
(Schachter H, Biochem Cell Biol 1986, 64:163-181).
[0004] Specific glycan structures regulate lymphocyte adhesion,
re-circulation and maturation as demonstrated by the GDP-fucose
deficiency in LADII patients (11), and immune defects associated
with C2 GlcNAc-T(L) (12) or ST3Gal-I (13) mutant mice. Depletion of
the .beta.1,6N-acetylglucosaminyltransferase V (Mgat5) modified
glycans by swainsonine, an inhibitor of .alpha.-mannosidase II,
potentiates antigen-dependent T cell proliferation, however, the
molecular basis of this effect is unknown (14). Mgat5 catalyzes the
addition of .beta.1,6GlcNAc to N-glycan intermediates found on
newly synthesized glycoproteins transiting the medial Golgi (15)
(FIG. 1A). The glycans are elongated in trans-Golgi to produce tri
(2,2,6) and tetra (2,4,2,6) antennary N-glycans which are
preferentially extended with N-acetyllactosamine
(Gal.beta.1,4GlcNAc) and polymeric forms of N-acetyllactosamine
also known as polylactosamine (6).
SUMMARY OF THE INVENTION
[0005] Applicants have demonstrated that differential receptor
glycosylation effects ligand-dependent clustering of receptors.
Applicants illustrated the effects of differential receptor
glycosylation with T cell receptors (TCR). T cell activation
requires clustering of a threshold number of T cell receptors (TCR)
at the site of antigen presentation, a number that is reduced by
CD28 co-receptor recruitment of signaling proteins to TCR (1-5).
Applicants demonstrate that a deficiency in
.beta.1,6N-acetylglucosaminyltransferase V (Mgat5), an enzyme in
the N-glycosylation pathway, lowers T cell activation thresholds by
directly enhancing TCR clustering. MGat5-deficient mice displayed
kidney autoimmune disease, enhanced delayed type hypersensitivity,
and increased susceptibility to experimental autoimmune
encephalomyelitis. Thus, dysregulation of Mgat5 in humans may
increase susceptibility to autoimmune diseases such as multiple
sclerosis.
[0006] Recruitment of TCR to agonist-coated beads, TCR signaling,
actin microfilament reorganization and agonist-induced
proliferation were enhanced in Mgat5.sup.-/- T cells. Mgat5
initiates GlcNAc .beta.1,6 branching on N-glycans, thereby
increasing N-acetyllactosamine (6), the lectin for galectins (7, 8)
proteins known to modulate T cell proliferation and apoptosis (9,
10). Indeed, galectin-3 was associated with the TCR complex at the
cell surface, an interaction dependent on Mgat5. Pre-treatment of
wild type T cells with lactose to compete for galectin binding
produced a phenocopy of Mgat5.sup.-/- TCR clustering. These data
indicate that a galectin-glycoprotein lattice strengthened by
Mgat5-modified glycans restricts TCR recruitment to the site of
antigen presentation.
[0007] In accordance with an aspect of the invention an isolated
complex is provided comprising one or more lectin (e.g. a galectin)
associated or interacting with a Mgat5 modified glycan or
polylactosamine modified glycan that is associated with receptor
clustering. The invention also provides a peptide derived from the
binding domain of a lectin, preferably a galectin, that interacts
with a Mgat5 modified glycan, or a polylactosamine modified glycan;
and, an oligosaccharide derived from a Mgat5 modified glycan or a
polylactosamine modified glycan that interacts with one or more
lectin (e.g. a galectin). The invention also contemplates
antibodies specific for these complexes, peptides, and
oligosaccharides.
[0008] The invention also contemplates an isolated lectin-Mgat5
modified glycan lattice comprising an array of multivalent
interactions among lectins, Mgat5 modified glycans, polylactosamine
modified glycans, and/or glycoproteins that are associated with
receptor clustering. The Mgt5 modified glycans and polylactosamine
modified glycans are preferably part of glycoproteins of receptors
including but not limited to TCR, growth factor receptors, and
cytokine receptors.
[0009] Still further the invention provides a method for evaluating
a test compound for its ability to regulate receptor clustering
through glycans on cell surfaces (e.g. through Mgat 5 modified
glyeans and/or polylactosamine modified glycans) comprising
assaying for alterations of the glycans in the presence of the test
compound. Alterations of the glycans may increase or enhance, or
inhibit or decrease receptor clustering thereby modifying signal
transduction by the receptors.
[0010] In an aspect of the invention, a method is provided for
evaluating a test compound for its ability to regulate receptor
clustering through a lectin-Mgat5 modified glycan lattice, in
particular a galectin-Mgat5 modified glycan lattice comprising
determining the effect of the test compound on the lattice or a
component thereof. A test compound may be a substance that
interacts with a component of a lectin-Mgat5 modified glycan
lattice. In particular, the substance may interact with a lectin
(e.g. galectin), Mgat5 modified glycan, or polylactosamine modified
glycan. The substance may be a molecule derived from a lectin (e.g.
galectin), Mgat5 modified glycan, polylactosamine modified glycan,
or lectin-Mgat5 modified glycan lattice; or, a substance which
inhibits or enhances the interaction of a lectin (e.g. galectin)
and a component of a lectin-Mgat5 modified glycan lattice (e.g. the
interaction of a galectin and Mgat5 modified glycan and/or
polylactosamine modified glycan).
[0011] In an embodiment, the method comprises (a) mixing a
galectin-Mgat5 modified glycan lattice, or a galectin and one or
more of a Mgat5 modified glycan and a polylactosamine modified
glycan, and a test compound, under conditions which maintain the
lattice or permit the formation of complexes between the galectin
and one or more glycan; and (b) removing and/or detecting
galectin-Mgat5 modified glycan lattice, complexes, galectin, Mgat5
modified glycan, or polylactosamine modified glycan.
[0012] The invention also encompasses the compounds identified
using methods of the invention.
[0013] The invention also contemplates cell-based assays. In an
aspect of the invention, a method is provided comprising (a)
providing cells with receptors whereby clustering of the receptors
is regulated through a lectin-Mgat5 modified glycan lattice or a
component thereof; (b) mixing the cells, lectin, and a test
compound under conditions which permit the formation of a
lectin-Mgat5 modified glycan lattice, complexes between a lectin
and one or more glycan of the lattice, and/or receptor clustering;
(c) detecting a lectin-Mgat5 modified glycan lattice, complexes,
lectin, Mgat5 modified glycan, polylactosamine modified glycan,
alterations to the lattice, complexes, lectin, Mgat5 modified
glycan, or polylactosamine modified glycan, or receptor clustering;
and (d) comparing to a control to determine if the test compound
alters the lectin-Mgat5 modified glycan lattice or component
thereof and potentially regulates receptor clustering.
[0014] Differential glycosylation of receptors has been found to
alter receptor clustering. Receptor clustering or oligomerization
is a requisite event for signal transduction of receptors,
including but not limited to receptors that stimulate immune
reactions, growth factor receptors, and cytokine receptors. Thus,
differences in glycans (e.g. Mgat5 modified glycans or
polylactosame modified glycans) on cell surfaces that are
associated with clustering of receptors including receptors that
stimulate immune reactions (e.g. T cell receptors, Ig receptors, B
cell receptors, NK receptors), the HER family of transmembrane
receptor tyrosine kinases [e.g. epidermal growth factor (EGF)
receptor also known as HER1 or Erb1, HER2 (neu, Erb2), HER3 (Erb3),
and HER4 (Erb4)], cadherin receptors (e.g. E-cadherin and
N-cadherin), interleukin (IL) receptors including IL-2 receptor,
TNF.gamma. receptor, and integrins, may affect clustering or
oligomerization of these receptors.
[0015] The invention provides a method for regulating receptor
clustering on cell surfaces comprising altering glycans on the cell
surface associated with receptor clustering. In an aspect the
invention provides a method for activating signal transduction in a
cell with receptors that cluster or oligomerize to thereby initiate
signal transduction comprising altering glycans associated with
clustering or oligomerization of the receptors.
[0016] Glycans can be altered by modulating one or more
glycosyltransferase enzyme involved in the synthesis of the
glycans, in particular N-glycans and N-glycan intermediates.
Altering glycans may involve increasing or decreasing Mgat5
modified glycans or polylactosamine modified glycans associated
with clustering of the receptors. In a preferred embodiment, an
enzyme involved in the synthesis of the glycans is modulated (e.g.
Mgat5).
[0017] In accordance with the present invention, a method is
provided for regulating receptor clustering on cell surfaces, in
particular ligand-dependent receptor clustering, more particularly
T cell receptor clustering, comprising modulating Mgat5 activity,
the amount of Mgat5 modified glycans, polylactosamine modified
glycans, or lectin-Mgat5 modified glycan lattice, or the amount of
binding or interaction of one or more components of a lectin-Mgat5
modified glycan lattice, (e.g. Mgat5 modified glycans or
polylactosamine modified glycans with lectins that bind to the
glycans, for example, galectins).
[0018] In accordance with another aspect of the invention, a method
is provided for treating or preventing a condition associated with
decreased or increased receptor clustering or a receptor clustering
defect in a subject comprising altering glycans associated with
receptor clustering. Glycans can be altered by modulating a
glycosyltransferase enzyme (e.g. Mgat5) involved in the synthesis
of the glycans.
[0019] In accordance with a particular aspect of the invention, a
method is provided for treating or preventing a condition
associated with decreased or increased receptor clustering (more
particularly T cell receptor clustering), or a receptor clustering
defect (more particularly a T cell receptor clustering defect),
comprising modulating Mgat5 activity, the amount of Mgat5 modified
glycans, polylactosamine modified glycans, or lectin-Mgat5 modified
glycan lattice, and/or the amount of binding or interaction of one
or more components of a lectin-MgatV modified glycan lattice (e.g.
a galectin, a Mgat5 modified glycan, polylactosamine modified
glycan, or glycoproteins).
[0020] The invention also contemplates compounds for regulating
receptor clustering. The compounds may be capable of directly or
indirectly modifying glycans involved in receptor clustering. Such
compounds may modulate the activity of an enzyme involved in the
synthesis of the glycans (e.g. a glycosyltransferase such as
Mgat5), the amount of the glycans, (e.g. the amount of Mgat5
modified glycans or polylactosamine glycans), and/or the amount of
binding of the glycans with a substance that binds to the glycans
thereby regulating receptor clustering (e.g. the binding or
interaction of Mgat5 modified glycans and galectins). The invention
also provides methods for assaying for such compounds. Compositions
comprising such compounds are also within the scope of the
invention.
[0021] In accordance with an aspect of the invention there is
provided a method of, and products for, diagnosing and monitoring
conditions characterized by an abnormality in clustering of a
receptor comprising assaying for differential glycosylation of the
receptor. Differential glycosylation may be assayed by determining
the presence of Mgat5 modified glycans, polylactosamine modified
glycans, lectin-Mgat5 modified glycan lattice, or an alteration or
change in such glycans or lattice, compared to a control.
[0022] The invention relates to the control of glycan-lectin
combinations (e.g. glacetin-polylactosamine modified glycan
lattice) identified using the invention.
[0023] These and other aspects, features, and advantages of the
present invention should be apparent to those skilled in the art
from the following drawings and detailed description.
DESCRIPTION OF THE DRAWINGS
[0024] The invention will now be described in relation to the
drawings in which:
[0025] FIG. 1 Immune phenotype in Mgat5.sup.-/- mice (A) Schematic
of the Golgi N-glycan biosynthesis pathway shows Mgat5 (TV) in the
production of a tetra (2,4,2,6) antennary (numbers in brackets
refer to the linkages of the antennae left to right). Abbreviations
are oligosaccharyltransferase, OT; the .alpha.-glucosidases, GI,
GII; the .beta.-N-acetylglycosaminyltransferases, TI, TII, TIV, TV
T(i); the .alpha.1,2mannosidases, MI, .alpha.1,3/6mannosidases MII,
MIII; .beta.1,4-galactosyltransferases, Gal-T;
.alpha.-sialyltransferases, ST; SW, position of swainsonine block.
The boxed structure
Gal.beta.1,4GlcNAc.beta.1,6(Gal.beta.1,4GlcNAc.beta.1,2)Mana binds
L-PHA. The galectin binding disaccharide N-acetyllactosamine
(Gal.beta.1,4GlcNAc) is present in all antennae, and units are
marked with brackets (}) in polylactosamine. (B) Distribution of
CD4+ and CD8+ cells in spleen and thymus by FACS analysis using
FITC- or phycoerythrin (PE)-conjugated antibodies (Pharmingen)
reactive to CD3.epsilon., CD4, and CD8. (C) TCR complex staining of
spleen cells by FITC-anti-CD3.epsilon. antibodies and FACS
analysis. (D) Light microscopy of kidney showing cresentic
glomerulonephritis with a large crescent (CR) of mononuclear cells
and fibrin obliterating the Bowman's space (BS) in Mgat5.sup.-/-
mice. (E) DTH inflammatory response in Mgat5.sup.-/- ( ) and
Mgat5.sup.+/+ (.quadrature.) mice exposed to oxazolone first on
their back, then 4 days later on the right ear. The results are
plotted as mean change.+-.S.E. in ear thickness relative to the
vehicle-treated left ear for 7 Mgat5.sup.-/- and 6 Mgat5.sup.+/+
control littermates. P<0.01 with a student t test comparing the
genotypes at 2-5 days.
[0026] FIG. 2 Mgat5.sup.-/- T cells are hypersensitive to TCR
agonists. (A) Spleen cells were cultured with anti
TCR.alpha./.beta. antibodies for 48 h. Filled circles,
Mgat5.sup.-/-; open squares, Mgat5.sup.+/+. (B) Purified T cells
from spleen were stimulated for 48 h with anti-CD3.epsilon.
antibody in the absence (.smallcircle.,.quadrature.) or presence (
, .box-solid.) of anti-CD28 antibody; Mgat5.sup.-/- (circles) and
Mgat5.sup.+/+ (squares) cells. (C) The Hill slope (n.sub.H) of the
sigmodal curves was calculated using
Y=x.sup.nH/(k.sup.nH+x.sup.nH). (D) Stimulation of splenic T cells
with PMA plus ionomycin for 48 h. (E) Stimulation of splenic T
cells from Mgat5.sup.-/- ( ) and Mgat5.sup.+/+ (.quadrature.) mice
with L-PHA and (F) Stimulation of splenic B cells with anti-IgM
antibody for 48 h. The means.+-.SD of triplicate determinations
were graphed.
[0027] FIG. 3 TCR clustering, actin reorganization and signaling in
T cells from Mgat5.sup.-/- and Mgat5.sup.+/+ mice. (A) TCR and
actin microfilament distribution in T cells stimulated by
anti-CD3.epsilon. coated beads. (B) Merged images of Mgat5.sup.-/-
and Mgat5.sup.+/+ cells. Clustering was observed in 5/6 and 0/6
randomly photographed cells, respectively. (C) TCR internalization
was monitored by FACS analysis using
FITC-anti-TCR.sub..alpha./.beta. antibodies to measure cell surface
TCR remaining at various times after the addition of
anti-CD3.epsilon. antibody. Changes in mean fluorescence intensity
(MFI) with time are graphed. T cells from Mgat5.sup.-/- (
,.diamond-solid.) or Mgat5.sup.+/+ (.quadrature.,*) mice were
treated with anti-CD3.epsilon. antibody ( ,.quadrature.); or with
PMA (.diamond-solid.,*). Similar results were obtained when the
stimulation and detection roles of anti-TCR.sub..alpha./.beta. and
anti-CD3 were reversed. (D) Actin polymer content in T cells from
Mgat5.sup.-/- ( ), or Mgat5.sup.+/+ (.quadrature.) mice at times
after stimulation with anti-CD3.epsilon. antibody, measured by
FACS. Western blot for phospho-Akt/PKB in T cell lysates following
addition of anti-CD.epsilon. antibody is shown. The values below
are fold increase in PKB-P normalized to PKB protein. (E) Ca.sup.2+
mobilization in purified T cells from Mgat5.sup.-/- ( ), and
Mgat5.sup.+/+ (.quadrature.) mice stimulated with anti-CD3.epsilon.
antibody. (F) Western blot with anti-phosphotyrosine antibody
detecting phosphorylated proteins in T cells lysates after
incubation with anti-CD3.epsilon. antibody coated beads for various
times. A longer exposure was used to reveal bands (arrowheads at
left) migrating as p95 and p36 shown below. Arrows at the right
indicate the positions of molecular mass markers. (G)
Immunoprecipitation of Zap70 and Western blotting for
phosphotyrosine (pY) to detect Zap70 and CD3.zeta. (values below
are CD3.zeta. ratio P23/P21).
[0028] FIG. 4 Lactose stimulates TCR aggregation and signaling in
Mgat5.sup.+/+. Purified T cells pre-incubated for 20 min with
buffer (A), 2 mM sucrose (B), or 2 mM lactose (C) then stimulated
with anti-CD3.epsilon. antibody-coated beads for 10 min, and
stained for TCR. Enhanced TCR clustering was observed in 0/10, 1/10
and 9/10, respectively. (D) Mgat5.sup.+/+ T cells incubated with
increasing concentrations of disaccharide (1/3 serial dilution from
2.4 mM) and stimulated for 1 min with anti-CD3.epsilon.
antibody-coated beads were compared for phosphotyrosine. Arrows at
the right indicate the positions of molecular mass markers. A
longer exposure of the lower molecular weight portion of the blot
is shown. (E) Galectin-3 detected by surface labeling with
NHS-biotin on T cells. Below, association of galectin-3 with
CD3.epsilon. and TCR.alpha. chain, and its disruption by Mgat5
deficiency and lactose is shown. (F) A model depicting restricted
mobility of TCR by interaction with a galectin-glycoprotein
network, which is stronger in Mgat5-expressing cells. (G) LacZ
activity in untreated (leftmost curve) and anti-CD3 and anti-CD28
stimulated (rightmost curve) T cells from Mgat5.sup.-/- mice. (H)
L-PHA binding to Mgat5.sup.+/+ T lymphocytes either untreated
(leftmost curve) or stimulated with anti-CD3 and anti-CD28 for 48 h
(rightmost curve).
DETAILED DESCRIPTION OF THE INVENTION
[0029] In accordance with the present invention there may be
employed conventional biochemistry, enzymology, molecular biology,
microbiology, and recombinant DNA techniques within the skill of
the art. Such techniques are explained fully in the literature. See
for example, Sambrook, Fritsch, & Maniatis, Molecular Cloning:
A Laboratory Manual, Second Edition (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A
Practical Approach, Volumes I and II (D. N. Glover ed. 1985);
Oligonucleotide Synthesis (M..J. Gait ed. 1984); Nucleic Acid
Hybridization B. D. Hames & S. J. Higgins eds. (1985);
Transcription and Translation B. D. Hames & S. J. Higgins eds
(1984); Animal Cell Culture R. I. Freshney, ed. (1986); Immobilized
Cells and enzymes IRL Press, (1986); and B. Perbal, A Practical
Guide to Molecular Cloning (1984).
Complexes, Peptides, and Oligosaccharides
[0030] In accordance with an aspect of the invention an isolated
complex is provided comprising one or more lectin associated or
interacting with a Mgat5 modified glycan, or polylactosamine
modified glycan that is associated with receptor clustering.
[0031] The term "isolated complex" refers to a complex
substantially free of cellular material or culture medium when
produced in vitro, or chemical reactants, or other chemicals when
chemically synthesized.
[0032] "Lectin" refers to a molecule that interacts with, binds, or
crosslinks carbohydrates. Preferably a lectin employed in the
present invention interacts with, binds, or crosslinks Mgat5
modified glycans polylactosamine modified glycans, and/or
glycoproteins. In an embodiment, the lectin is a galactose-binding
protein, preferably a galectin.
[0033] "Galectin" refers to a member of the galectin family of
beta-galactoside-binding proteins (see "Galectins: A Family of
Animal beta-Galactoside-Binding Lectins" (1994) by S. H. Barondes,
V. Castronovo, D. N. W. Cooper, R. D. Cummings, K. Drickamer, et
al., In Cell 76, 597-598). Galectins includes lectins that bind
beta-galactoside carbohydrate moieties in a thiol-dependent manner.
(Reviewed in Hadari, Y. R. et al. (1998) J. Biol. Chem.
270:3447-3453.) Galectins are widely expressed and developmentally
regulated. Galectins contain a characteristic carbohydrate
recognition domain (CRD). The CRD is about 140 amino acids and
contains several stretches of about 1-10 amino acids that are
highly conserved among all galectins. Examples of galectins are
galectin-1 through -10. In preferred embodiments of the invention,
the galectin is galectin-3. Galectin-3 has one CRD, a short
N-terminal domain and an intervening proline, glycine and
tyrosine-rich domain which consists of repeats of 7-10 conserved
amino acids. A "galectin" may be a monomer, dimer, or tetramer,
preferably a dimer.
[0034] "Glycosyltransferase" refers to an enzyme involved in the
synthesis of glycans, preferably the synthesis of N-glycans or
O-glycans, more preferably N-glycans, most preferably tri (2,2,6)
and tetra (2,4,2,6) antennary N-glycans, which are preferentially
extended with N-acetyllactosamine and polylactosamine (i.e.
polylactosamine modified glycan). Examples of such
glycosyltransferase enzymes are Mgat5, core 2 GlcNAc transferase,
GlcNAcT(i), and .beta.1-4 galactosyltransferase. The term
"glycosyltransferase" includes a wild type enzyme, or part thereof,
or a mutant, variant or homolog of such an enzyme.
[0035] "Mgat5" refers to .beta.1,6N-acetylglucosaminyltransferase V
enzymes, preferably mammalian enzymes, that catalyze the addition
of N-acetylglucosamine in beta 1-6 linkage to the alpha-linked
mannose of biantennary N-linked oligosaccharides. Examples of Mgat5
enzymes are found on the ExPASy.RTM. proteomics server as Enzyme:
2.4.1.155, and include human Mgat5 (Saito et al, 1994; gb:d17716,
sw:q09328), and rat Mgat5 (Shoreibah et al 1993, J. Biol. Chem.
268: 15381-15385; gb114284, sw:q08834). "Mgat5" includes the wild
type enzyme, or part thereof, or a mutant, variant or homolog of
such an enzyme.
[0036] The term "wild type" refers to a polypeptide having a
primary amino acid sequence which is identical with the native
enzyme (for example, the human or mouse enzyme). The term "mutant"
refers to a polypeptide having a primary amino acid sequence which
differs from the wild type sequence by one or more amino acid
additions, substitutions or deletions. Preferably, the mutant has
at least 90% sequence identity with the wild type sequence.
Preferably, the mutant has 20 mutations or less over the whole
wild-type sequence. More preferably the mutant has 10 mutations or
less, most preferably 5 mutations or less over the whole wild-type
sequence.
[0037] The term "variant" refers to a naturally occurring
polypeptide that differs from a wild-type sequence. A variant may
be found within the same species (i.e. if there is more than one
isoform of the enzyme) or may be found within a different species.
Preferably the variant has at least 90% sequence identity with the
wild type sequence. Preferably, the variant has 20 mutations or
less over the whole wild-type sequence. More preferably, the
variant has 10 mutations or less, most preferably 5 mutations or
less over the whole wild-type sequence.
[0038] The term "part" indicates that the polypeptide comprises a
fraction of the wild-type amino acid sequence. It may comprise one
or more large contiguous sections of sequence or a plurality of
small sections. The polypeptide may also comprise other elements of
sequence, for example, it may be a fusion protein with another
protein (such as one which aids isolation or crystallization of the
polypeptide). Preferably the polypeptide comprises at least 50%,
more preferably at least 65%, most preferably at least 80% of the
wild-type sequence.
[0039] The term "homolog" means a polypeptide having a degree of
homology with the wild-type amino acid sequence. The term
"homology" refers to a degree of complementarity. There may be
partial homology or complete homology. In an embodiment of the
invention a glycosyltransferase, in particular Mgat5, is
substantially homologous to a wild type enzyme. A sequence that is
"substantially homologous" refers to a partially complementary
sequence that at least partially inhibits an identical sequence
from hybridizing to a target nucleic acid. Inhibition of
hybridization of a completely complementary sequence to the target
sequence may be examined using a hybridization assay (e.g. Southern
or northern blot, solution hybridization, etc.) under conditions of
reduced stringency. A sequence that is substantially homologous or
a hybridization probe will compete for and inhibit the binding of a
completely homologous sequence to the target sequence under
conditions of reduced stringency. However, conditions of reduced
stringency can be such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested
using a second target sequence which lacks even a partial degree of
complementarity (e.g., less than about 30% homology or identity).
The substantially homologous sequence or probe will not hybridize
to the second non-complementary target sequence in the absence of
non-specific binding.
[0040] A sequence of an enzyme contemplated by the invention may
have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
identity. The phrases "percent identity" or "% identity" refer to
the percentage comparison of two or more amino acid or nucleic acid
sequences. Percent identity can be determined electronically using
for example the MegAlign.TM. program (DNASTAR, Inc., Madison Wis.).
The MegAlign.TM. program can create alignments between two or more
sequences according to different methods, e.g., the Clustal method.
(See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.)
Percent identity between nucleic acid sequences can also be
determined by other methods known in the art, e.g., the Jotun Hein
method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)
In addition, identity between sequences can be determined by other
methods known in the art, e.g., by varying hybridization
conditions.
[0041] "Mgat5 modified glycan" refers to a
GlcNAc.beta.1,6Man.alpha.1,6-branched N-glycan structure. The
glycans are produced by Mgat5 which catalyzes the addition of
.beta.1,6GlcNAc to N-glycan intermediates found on newly
synthesized glycoproteins transiting the medial Golgi (15). The
glycans are elongated in trans-Golgi to produce tri (2,2,6) and
tetra (2,4,2,6) antennary N-glycans. A Mgat5 modified glycan may be
substituted with for example polylactosamine (i.e. it may be a
polylactosamine modified glycan). A Mgat5 modified glycan may be
part of or covalently linked to a cell surface glycoprotein,
including a glycoprotein of the T cell receptor complex.
[0042] "Polylactosamine modified glycan" refers to specific glycan
structures comprising N-acetyllactosamine (Gal.beta.1,4GlcNAc) and
polymeric forms of N-acetyllactosamine, also known as poly
N-acetyllactosamine or polylactosamine (6). Preferably the
polylactosamine modified glycan is an Mgat5 modified glycan
substituted with poly N-acetyllactosamine. A polylactosamine
modified glycan may be part of or covalently linked to a cell
surface glycoprotein, including a glycoprotein of the T cell
receptor complex.
[0043] The invention also contemplates a lectin-Mgat5 modified
glycan lattice, preferably a galectin-Mgat5 modified glycan
lattice.
[0044] A "lattice" is an arrangement of multiple interacting
molecules, in particular, an arrangement or array of mulitvalent
interactions among lectins, glycans, and/or glycoproteins. A
preferred lattice of the invention is a lectin-Mgat5 modified
glycan lattice.
[0045] A "lectin-Mgat5 modified glycan lattice" refers to a lattice
formed from the multivalent interactions of lectins and Mgat5
modified glycans, polylactosamine modified glycans, and/or
glycoproteins that are associated with receptor clustering. When
the lectin is a galectin the lattice is referred to as a
"galectin-Mgat5 modified glycoprotein lattice". The stoichiometry
of components of a lattice preferably provides optimal occupation
of the lectin-glycan binding sites to create strong interactions
among the components of the lattice resulting in an impediment to
receptor clustering.
[0046] A lectin-Mgat5 modified glycan lattice, in particular a
galectin-Mgat5 modified glycan lattice may restrict clustering of
receptors on cell surfaces. By way of example, the galectin-Mgat
modified glycan lattice may restrict TCR recruitment to the site of
antigen presentation.
[0047] The invention also provides a peptide derived from the
binding domain or binding site of a lectin (e.g. a carbohydrate
recognition domain of a galectin) that interacts with a glycan
component of a lectin-Mgat5 modified glycan lattice (e.g. a Mgat5
modified glycan, a polylactosamine modified glycan, or
glycoprotein); or, an oligosaccharide derived from a Mgat5 modified
glycan or polylactosamine modified glycan of a lectin-Mgat5
modified glycan lattice that interacts with one or more lectin, in
particular a galectin. The peptide may preferably be derived from a
carbohydrate recognition domain of a galectin.
[0048] The invention also relates to an oligosaccharide derived
from a Mgat5 modified glycan or polylactosamine modified glycan,
preferably of a T cell receptor, that interacts with one or more
galectin.
[0049] By being "derived from" is meant any molecular entity which
is identical or substantially equivalent to the native binding site
of a molecule in a complex, or lattice of the invention (e.g. a
lectin in particular, a galectin, or a glycan, in particular a
Mgat5 modified glycan or polylactosamine modified glycan). A
peptide or oligosaccharide derived from a specific binding site may
encompass the amino acid or carbohydrate sequence of a naturally
occurring binding site, any portion of that binding site, or other
molecular entity that functions to bind to an associated or
interacting molecule. A peptide or oligosaccharide derived from
such a binding domain will interact directly or indirectly with an
associated or interacting molecule in such a way as to mimic the
native binding site. Such peptides and oligosaccharides may include
competitive inhibitors, peptide mimetics, and the like.
[0050] The term "interact", "interacting", or "interaction" refers
to a stable association between two molecules due to, for example,
electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions
under physiological conditions.
[0051] "Peptide mimetics" are structures which serve as substitutes
for peptides in interactions between molecules (See Morgan et al
(1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide
mimetics include synthetic structures that may or may not contain
amino acids and/or peptide bonds but retain the structural and
functional features of a peptide, or enhancer or inhibitor of the
invention. Peptide mimetics also include peptoids, oligopeptoids
(Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide
libraries containing peptides of a designed length representing all
possible sequences of amino acids corresponding to a peptide, or
enhancer or inhibitor of the invention.
[0052] The invention also contemplates an altered glycan of a cell
surface glycoprotein associated with receptor clustering resulting
from the inhibition of a glycosyltransferase involved in the
synthesis of the glycan. In an embodiment the altered glycan is an
altered Mgat5 modified glycan or an altered polylactosamine
modified glycan. By way of example, an altered Mgat5 modified
glycan has a deficiency of .beta.1-6 branches, and an altered
polylactosamine modified glycan has a deficiency of
N-acetyllactosamine or polylactosamine. An altered Mgat5 modified
glycan or altered polylactosamine modified glycan cannot
substantially interact or associate with a lectin, preferably a
galectin.
[0053] Mgat5 modified glycans, polylactosoamine modified glycans
and altered glycans may be assayed using substances that bind to
the glycans. The substances that bind to the glycans may be
antibodies or lectins. For example, leukoagglutinin (L-PHA) is a
tetravalent plant lectin that binds specifically to Mgat5 modified
glycans.
[0054] The invention contemplates antibodies specific for the
complexes, lattice, peptides, oligosaccharides, and altered glycans
of the invention. Antibodies include intact monoclonal or
polyclonal antibodies, and immunologically active fragments (e.g. a
Fab, (Fab).sub.2 fragment, or Fab expression library fragments and
epitope-binding fragments thereof), an antibody heavy chain, and
antibody light chain, a genetically engineered single chain Fv
molecule (Ladner et al, U.S. Pat. No. 4,946,778), humanized
antibodies, or a chimeric antibody, for example, an antibody which
contains the binding specificity of a murine antibody, but in which
the remaining portions are of human origin. Antibodies including
monoclonal and polyclonal antibodies, fragments and chimeras, may
be prepared using methods known to those skilled in the art.
[0055] Antibodies specific for a Mgat5 modified glycan,
polylactosamine modified glycan, complex, lattice, or an altered
glycan may be produced in Mgat5.sup.-/- mice using conventional
methods.
[0056] Antibodies specific for the complexes, lattice, peptides,
oligosaccharides, and altered glycans of the invention may be used
to detect the complexes, lattice, etc. in tissues and to determine
their tissue distribution. In vitro and in situ detection methods
using the antibodies of the invention may be used to assist in the
prognostic and/or diagnostic evaluation of disorders mediated by or
involving receptor clustering, more particularly T cell receptor
mediated disorders. Antibodies specific for the complexes, lattice,
etc. of the invention may also be used therapeutically to modulate
receptor clustering, more particularly T cell receptor clustering
(i.e. T cell activation).
Evaluating Compounds that Regulate Receptor Clustering
[0057] The invention provides a method for evaluating a test
compound for its ability to effect or regulate receptor clustering
through glycans on cell surfaces (e.g. glycans of the receptor such
as Mgat5 modified glycans or polylactosamine modified glycans).
Changes to glycans on cell surfaces may increase or decrease
receptor clustering thereby modifying signal transduction by the
receptors.
[0058] "Receptor clustering" or "clustering of receptors" refers to
the association of one or more receptor molecules on the surface of
a cell to thereby initiate signal transduction, endocytosis, and
others events in the cell. Receptor clustering may be initiated or
induced by the interaction of ligands or anti-receptor antibodies
with receptor molecules. Thus, in an aspect of the invention
receptor clustering is ligand-dependent. Examples of receptor
molecules include but are not limited to receptors that stimulate
immune reactions (e.g. T cell receptors, Ig receptors, B cell
receptors, and NK receptors), members of the HER family of
transmembrane receptor tyrosine kinases [e.g. epidermal growth
factor (EGF) receptor also known as HER1 or Erb1, HER2 (neu, Erb2),
HER3 (Erb3), and HER4 (Erb4)], cadherin receptors (e.g. E-cadherin
and N-cadherin), interleukin (IL) receptors including IL-2
receptor, TNF.gamma. receptor, and integrins. In an embodiment, T
cell receptor clustering is down regulated by a lectin-Mgat5
modified glycan lattice which slows the migration of T cell
receptors into clusters at the immune synapse. Dissociation of the
lectin and glycan(s) of the lattice enhances T cell receptor
clustering lowering the T cell activation threshold.
[0059] An aspect of the invention provides a method for evaluating
a test compound for its ability to regulate receptor clustering
through a lectin-Mgat5 modified glycan lattice, in particular a
galectin-Mgat5 modified glycan lattice, or a component thereof.
[0060] Methods of the invention are designed to identify compounds
or substances that affect receptor clustering particularly T cell
receptor clustering (i.e. T cell activation). Novel substances are
therefore contemplated that bind to or interact with molecules in a
complex or lattice, or bind to other molecules that interact with
the molecules in the complex or lattice, to compounds that
interfere with, or enhance the interaction of the molecules in a
complex or lattice, or other compounds that interact with the
molecules. Therefore, by way of example, a compound may be a
substance that binds to a lectin (e.g. galectin), a polylactosamine
modified glycan, or a Mgat5 modified glycan; a molecule derived
from a lectin (e.g.galectin), Mgat5 modified glycan, or
polylactosamine modified glycan; or a substance which inhibits or
enhances the interaction of a lectin (e.g. galectin) and a Mgat5
modified glycan or a polylactosamine modified glycan.
[0061] A compound that enhances or inhibits the interaction of a
lectin (e.g. galectin) and a Mgat5 modified glycan or
polylactosamine modified glycan is intended to include a peptide or
peptide fragment derived from the binding site of a lectin (e.g.
galectin), or oligosaccharide or fragment thereof derived from the
binding site of the Mgat5 modified glycan or polylactosamine
modified glycan. An enhancer or inhibitor will not include the full
length sequence of the wild-type molecule. Peptide mimetics,
oligosaccharide mimetics, and synthetic molecules with physical
structures designed to mimic structural features of particular
peptides or oligosaccharides, may serve as inhibitors or enhancers.
Inhibitors or enhancers may affect receptor clustering, in
particular T-cell receptor clustering. The enhancement or
inhibition may be direct, or indirect, or by a competitive or
non-competitive mechanism.
[0062] The substances identified using the methods of the invention
include but are not limited to peptides such as soluble peptides
including Ig-tailed fusion peptides, members of random peptide
libraries and combinatorial chemistry-derived molecular libraries
made of D- and/or L-configuration amino acids, phosphopeptides
(including members of random or partially degenerate, directed
phosphopeptide libraries), oligosaccharides, antibodies [e.g.
polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single
chain antibodies, fragments, (e.g. Fab, F(ab).sub.2, and Fab
expression library fragments, and epitope-binding fragments
thereof)], and small organic or inorganic molecules. The substance
or compound may be an endogenous physiological compound or it may
be a natural or synthetic compound.
[0063] The invention particularly contemplates a method for
evaluating a compound for its ability to modulate the biological
activity of a complex or lattice of the invention, by assaying for
an agonist or antagonist (i.e. enhancer or inhibitor) of the
binding or interaction of molecules in the complex or lattice. The
basic method for evaluating if a compound is an agonist or
antagonist of the binding of molecules in a complex or lattice of
the invention, is to prepare a reaction mixture containing the
molecules and the substance under conditions which permit the
formation of complexes or a lattice, in the presence of a test
compound. The test compound may be initially added to the mixture,
or may be added subsequent to the addition of molecules. Control
reaction mixtures without the test compound or with a placebo are
also prepared. The formation of complexes or a lattice is detected
and the formation of complexes or a lattice in the control reaction
but not in the reaction mixture indicates that the test compound
interferes with the interaction of the molecules. The reactions may
be carried out in the liquid phase or the molecules, or test
compound may be immobilized as described herein.
[0064] It will be understood that the agonists and antagonists i.e.
inhibitors and enhancers that can be assayed using the methods of
the invention may act on one or more of the binding sites on the
interacting molecules in the complex or lattice including agonist
binding sites, competitive antagonist binding sites,
non-competitive antagonist binding sites or allosteric sites.
[0065] The invention also makes it possible to screen for
antagonists that inhibit the effects of an agonist of the
interaction of molecules in a complex or lattice of the invention.
Thus, the invention may be used to assay for a compound that
competes for the same binding site of a molecule in a complex or
lattice of the invention.
[0066] In an embodiment, the method comprises mixing a lectin-Mgat5
modified glycan lattice or a component thereof (e.g. lectin such as
a galectin, a Mgat5 modified glycan, polylactosamine modified
glycan), and a test compound under conditions which maintain the
lattice or permit the formation of complexes between the lectin and
one or more of the Mgat5 modified glycan, and polylactosamine
modified glycan, and removing and/or detecting lectin-Mgat5
modified glycan lattice, complexes, lectin, Mgat5 modified glycan,
or polylactosamine modified glycan. The invention also encompasses
the compounds identified using this method of the invention.
[0067] Substances which modulate the activity of a complex or
lattice of the invention can be identified based on their ability
to bind to a molecule in a complex or lattice of the invention.
Therefore, the invention also provides methods for identifying
novel substances which bind molecules in a complex or lattice of
the invention. Substances identified using the methods of the
invention may be isolated, cloned and sequenced using conventional
techniques.
[0068] Novel substances which can bind with a molecule in a complex
or lattice of the invention may be identified by reacting one of
the molecules with a test substance which potentially binds to the
molecule, under conditions which permit the formation of
substance-molecule conjugates and removing and/or detecting the
conjugates. The conjugates can be detected by assaying for
substance-molecule conjugates, for free substance, or for
non-complexed molecules. Conditions which permit the formation of
substance-molecule conjugates may be selected having regard to
factors such as the nature and amounts of the substance and the
molecule.
[0069] The substance-molecule conjugate, free substance or
non-complexed molecules may be isolated by conventional isolation
techniques, for example, salting out, chromatography,
electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combinations
thereof. To facilitate the assay of the components, antibody
against the molecule or the substance, or labelled molecule, or a
labelled substance may be utilized. The antibodies, proteins, or
substances may be labelled with a detectable substance as described
above.
[0070] A molecule, complex, or lattice of the invention, or a
substance or compound used in a method of the invention may be
insolubilized. For example, a molecule, complex etc. may be bound
to a suitable carrier such as agarose, cellulose, dextran,
Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter
paper, ion-exchange resin, plastic film, plastic tube, glass beads,
polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid
copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The
carrier may be in the shape of, for example, a tube, test plate,
beads, disc, sphere etc. The insolubilized molecule, complex etc.
may be prepared by reacting the material with a suitable insoluble
carrier using known chemical or physical methods, for example,
cyanogen bromide coupling.
[0071] Compounds that bind to molecules of a complex or lattice of
the invention or that interact with a molecule that binds to a
molecule of a complex or lattice of the invention may be assayed by
identifying protein-protein or protein-carbohydrate interactions
using conventional methods such as co-immunoprecipitation,
crosslinking and co-purification through gradients or
chromatographic columns. Methods may also be employed that result
in the simultaneous identification of genes which encode proteins
interacting with a molecule. These methods include probing
expression libraries with labeled molecules. Additionally, x-ray
crystallographic studies may be used as a means of evaluating
interactions with substances and molecules. For example, purified
recombinant molecules in a complex of the invention when
crystallized in a suitable form are amenable to detection of
intra-molecular interactions by x-ray crystallography. Spectroscopy
may also be used to detect interactions and in particular,
Q-TOF.RTM. instrumentation may be used. Two-hybrid systems may also
be used to detect protein interactions in vivo.
[0072] It will also be appreciated that the complexes or lattices
of the invention may be reconstituted in vitro and the effect of a
test substance may be evaluated in the reconstituted system.
[0073] The invention also contemplates cell based assays. In an
aspect of the invention, a method is provided comprising (a)
providing cells with receptors whereby clustering of the receptors
is regulated through a lectin-Mgat5 modified glycan lattice or
component thereof (e.g. lectin, Mgat5 modified glycans, and
polylactosamine modified glycans); (b) mixing the cells, lectin,
and a test compound, under conditions which permit the formation of
a lectin-Mgat5 modified glycan lattice, complexes between a lectin
and one or more glycan of the lattice, and/or receptor clustering;
(c) detecting a lectin-Mgat5 modified glycan lattice, complexes,
lectin, Mgat5 modified glycan, polylactosamine modified glycan,
alterations to the lattice, complexes, lectin, Mgat5 modified
glycan, or polylactosamine modified glycan, or detecting receptor
clustering; and (d) comparing to a control to determine if the test
compound potentially regulates receptor clustering.
[0074] In another aspect of the invention, a method is provided
comprising (a) providing cells with receptors whereby clustering of
the receptors is regulated through a lectin-Mgat5 modified glycan
lattice or component thereof (e.g. lectin, Mgat5 modified glycans,
and polylactosamine modified glycans); (b) mixing the cells,
lectin, a test compound, and a ligand for the receptor that induces
receptor clustering, under conditions which permit receptor
clustering; (c) detecting receptor clustering; and (d) comparing to
a control to determine if the test compound potentially regulates
receptor clustering. The lattice on the cell surface may regulate
the threshold cooperativity and dynamic range of ligand dependent
responses.
[0075] In an embodiment of the invention a method is provided which
comprises; [0076] (a) mixing cells with T cell receptors comprising
Mgat5 modified glycans or polylactosamine modified glycans, one or
more galectin, and a test compound under conditions suitable for
producing a galectin-Mgat5 modified glycan lattice; [0077] (b)
assaying for a galectin-Mgat5 modified glycan lattice; and [0078]
(c) comparing to a control in the absence of the test compound to
determine if the test compound has the potential to regulate
receptor clustering.
[0079] In an embodiment of the invention a method is provided which
comprises; [0080] (a) mixing cells with T cell receptors and Mgat5
modified glycans and/or polylactosamine modified glycans on their
surface, one or more galectin, and a test compound under conditions
suitable for producing receptor clustering of the T cell receptors;
[0081] (b) assaying for T cell receptor clustering or T cell
signaling or activation; [0082] (c) comparing to a control to
determine if the test compound has the potential for regulating
receptor clustering.
[0083] In a further embodiment of the invention, a method is
providing for evaluating a test compound for its potential to
regulate receptor clustering comprising: [0084] (a) mixing cells
with T cell receptors and Mgat5 modified glycans or polylactosamine
modified glycans on their surface, one or more galectin, and a test
compound under conditions suitable for producing a galectin-Mgat5
modified glycan lattice; [0085] (b) assaying for Mgat5 modified
glycans, polylactosamine modified glycans, galectin, or a
galectin-Mgat5 modified glycan lattice, or alterations to the
glycans or lattice; [0086] (c) comparing to a control where an
alteration to a Mgat5 modified glycan, polylactosamine modified
glycan, galectin, or a galectin-Mgat5 modified glycan lattice,
indicates that the test compound has potential to regulate receptor
clustering.
[0087] In a still further embodiment of the invention, a method is
providing for evaluating a test compound for its potential to
regulate receptor clustering comprising: [0088] (a) mixing
Mgat5.sup.-/- T cells and a test compound under conditions suitable
for clustering of T cell receptors on the T cells; [0089] (b)
assaying for T cell receptor clustering or T cell signaling; and
[0090] (c) comparing to a control to determine if the test compound
has potential to regulate receptor clustering.
[0091] In a still further embodiment of the invention, a method is
providing for evaluating a test compound for its potential to
regulate receptor clustering comprising: [0092] (a) mixing
Mgat5.sup.-/- T cells, one or more galectin, and a test compound
under conditions suitable for producing a galectin-Mgat5 modified
glycan lattice; [0093] (b) assaying for galectin-Mgat5 modified
glycan lattice or T cell receptor clustering or T cell signaling;
and [0094] (c) comparing to a control to determine if the test
compound has potential to regulate receptor clustering.
[0095] The methods for evaluating a test compound for potential to
regulate T cell receptor clustering may include an antigen
presenting cell, or a bead coated with an antigen or anti-TCR
antibody may be used to induce or initiate T cell receptor
clustering.
[0096] Mgat5 modified glycans, polylactosoamine modified glycans,
and lectin-Mgat5 modified glycan lattices may be assayed in the
methods of the invention using substances that bind to Mgat5
modified glycans, polylactosoamine modified glycans, or the
lattices. The substances that bind to the glycans and lattices may
be antibodies or lectins. For example, leukoagglutinin (L-PHA) may
be used to assay for Mgat5 modified glycans.
[0097] T cell receptor clustering or T cell signaling or activation
may be assayed using the methods illustrated herein and other
standard methods known to a skilled artisan.
[0098] The invention contemplates methods for assaying for
compounds and substances that regulate receptor clustering by
modulating the activity of one or more enzyme involved in the
biosynthesis of Mgat5 modified glycans or polylactosamine modified
glycans, in particular a glycosyltransferase, more particularly
Mgat5. Examples of methods for screening for substances that
modulate the activity of such enzymes are illustrated herein for
Mgat5. The invention also contemplates methods for screening for
compounds and substances that modulate the amount of Mgat5 modified
glycans or polylactosamine modified glycans.
[0099] Therefore, the invention provides methods for screening for
substances having potential pharmaceutical utility in the treatment
and diagnosis of conditions associated with increased or decreased
receptor clustering, particularly T cell receptor clustering. In an
embodiment of the invention a method of assaying for a therapeutic
is provided comprising assaying for a substance that inhibits or
stimulates the activity of Mgat5. Substances that inhibit or
stimulate Mgat5 activity may be identified by reacting Mgat5 with
an acceptor substrate and a sugar donor in the presence of a
substance suspected of inhibiting Mgat5, under conditions whereby
the Mgat5 is capable of transferring the sugar donor to the
acceptor substrate to produce a sugar donor-acceptor substrate
complex, and determining the effect of the substance by assaying
for sugar donor-acceptor substrate complexes, unreacted Mgat5,
unreacted sugar nucleotide donor or unreacted acceptor
substrate.
[0100] Suitable acceptor substrates include a saccharide,
oligosaccharides, polysaccharides, glycopeptides, glycoproteins, or
glycolipids which are either synthetic with linkers at the reducing
end or naturally occurring structures, for example,
asialo-agalacto-fetuin glycopeptide. The sugar donor may be a
nucleotide sugar, dolichol-phosphate-sugar or
dolichol-pyrophosphate-oligosaccharide, for example, uridine
diphospho-N-acetylglucosamine (UDP-GlcNAc), or derivatives or
analogs thereof.
[0101] The Mgat5 may be obtained from commercial sources; it may be
purified from immortalized cell lines such as small cell lung
cancer cells such as QG (Gu, J. et al. J. Biochem. 113, 111-116,
1993); or prepared by expression of the gene encoding Mgat5 in host
cells.
[0102] The acceptor substrate or sugar donor may be labeled with a
detectable substance, and the interaction of the enzyme with the
acceptor and sugar donor will give rise to a detectable change. The
detectable change may be colorimetric, photometric, radiometric,
potentiometric, etc. The activity of Mgat5 may also be determined
using methods based on HPLC (Koenderman et al., FEBS Lett. 222:42,
1987) or methods employed synthetic oligosaccharide acceptors
attached to hydrophobic aglycones (Palcic et al Glycoconjugate
5:49, 1988; and Pierce et al, Biochem. Biophys. Res. Comm. 146:
679, 1987).
[0103] The Mgat5 is reacted with the acceptor substrate and sugar
donor at a pH and temperature and in the presence of a metal
cofactor, usually a divalent cation like manganese, effective for
the enzyme to transfer the sugar donor to the acceptor substrate,
and where one of the components is labeled, to produce a detectable
change. It is preferred to use a buffer with the acceptor substrate
and sugar donor to maintain the pH within the pH range effective
for the proteins. The buffer, acceptor substrate, and sugar donor
may be used as an assay composition. Other compounds such as EDTA
and detergents may be added to the assay composition.
[0104] Substances that inhibit or stimulate Mgat5 activity may also
be assayed by treating immortalized cells that express Mgat5 with a
substance suspected of inhibiting or stimulating Mgat5, and
comparing the morphology of the cells with the morphology of the
cells in the absence of the substance and/or with immortalized
cells that do not express Mgat5.
[0105] Still further, a substance that inhibits or stimulates Mgat5
activity may also be identified by treating a cell that expresses
Mgat5 with a substance that is suspected of affecting Mgat5
activity, and assaying for Mgat5-modified glycans or
polylactosamine modified glycans on the surface of the cell.
Mgat5-modified glycans and polylactosamine modified glycans can be
measured using methods described herein and known in the art. For
example, cells expressing Mgat5-modified glycans may be treated
with a substance suspected of inhibiting or stimulating
Mgat5-modified glycans. A lectin such as L-PHA is then added and
the amount of binding can be compared to control cells which have
not been treated with the substance and/or which do not express
Mgat5-modified glycans.
[0106] In another method of the invention, immortalized cells
expressing Mgat5-modified glycans may be treated with a substance
suspected of inhibiting or stimulating Mgat5. The cells can be
treated with a lectin such as L-PHA and the sensitivity to the
lectin can be compared with controls cells which have not been
treated with the substance and/or which do not express Mgat5.
Examples of immortalized cells which can be used in the method are
immortalized lung epithelial cell lines such as CHO cells, Mv1Lu
cells, MDAY-D2 lymphoma, and lectin-resistant variants of these
cell lines, which are transfected with a Mgat5 vector and MDCK
cells. In the absence of an inhibitor the cells should show signs
of morphologic transformation. In particular, morphologic
transformation is evidenced by (a) fibroblastic morphology, spindle
shape and pile up; (b) the cells are less adhesive to substratum;
(c) there is less cell-cell contact in monolayer culture; (d) there
is reduced growth-factor requirements for survival and
proliferation; (e) the cells grow in soft-agar or other semi-solid
medium; (f) there is a lack of contact inhibition and increased
apoptosis in low-serum high density cultures; (g) there is enhanced
cell motility; and, (h) there is invasion into extracellular matrix
and secretion of proteases. Substances which interfere with one or
more of these phenotypes may be considered to inhibit Mgat5.
[0107] Substances which inhibit or stimulate transcription or
translation of a gene encoding a glycosyltransferase in particular
Mgat5 may be identified by transfecting a cell with an expression
vector comprising a recombinant molecule containing a nucleic acid
sequence encoding the glycosyltransferase (e.g. Mgat5), the
necessary elements for the transcription or translation of the
nucleic acid sequence and a reporter gene, in the presence of a
substance suspected of inhibiting or stimulating transcription or
translation of the gene encoding the glycosyltransferase (e.g.
Mgat5), and comparing the level of expression of the
glycosyltransferase (e.g. Mgat5) or the expression of the protein
encoded by the reporter gene with a control cell transfected with
the nucleic acid molecule in the absence of the substance. The
method can be used to identify transcription and translation
inhibitors or stimulators of the gene encoding the
glycosyltransferase (e.g. Mgat5).
[0108] The nucleic acid molecule encoding the glycosyltransferase
may be constructed having regard to the sequence of the
glycosyltransferase gene (e.g. see Saito et al., 1994 supra for
Mgat5 gene sequence) using chemical synthesis and enzymatic
ligation reactions following procedures known in the art.
[0109] Suitable transcription and translation elements may be
derived from a variety of sources, including bacterial, fungal,
viral, mammalian, or insect genes. Selection of appropriate
transcription and translation elements is dependent on the host
cell chosen, and may be readily accomplished by one of ordinary
skill in the art. Examples of such elements include: a
transcriptional promoter and enhancer, an RNA polymerase binding
sequence, a ribosomal binding sequence, including a translation
initiation signal. Additionally, depending on the host cell chosen
and the vector employed, other genetic elements, such as an origin
of replication, additional DNA restriction sites, enhancers, and
sequences conferring inducibility of transcription may be
incorporated into the expression vector. It will also be
appreciated that the necessary transcription and translation
elements may be supplied by the native gene and/or its flanking
sequences.
[0110] Examples of reporter genes are genes encoding a protein such
as .beta.-galactosidase (e.g. lac Z), chloramphenicol,
acetyl-transferase, firefly luciferase, or an immunoglobulin or
portion thereof such as the Fc portion of an immunoglobulin
preferably IgG. Transcription of the reporter gene is monitored by
changes in the concentration of the reporter protein such as
.beta.-galactosidase, chloramphenicol acetyltransferase, or firefly
luciferase. This makes it possible to visualize and assay for
expression of recombinant molecules to determine the effect of a
substance on expression of the glycosyltransferase (e.g. Mgat5)
gene.
[0111] Mammalian cells suitable for carrying out the present
invention include any malignant cells, for example, COS (e.g., ATCC
No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No.
CCL 61), HeLa (e.g., ATCC No. CCL 2), and 293 (ATCC No. 1573).
Suitable expression vectors for directing expression in mammalian
cells generally include a promoter. Common promoters include SV40,
MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early,
immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.
[0112] Protocols for the transfection of mammalian cells are well
known in the art and include calcium phosphate mediated
electroporation, retroviral, and protoplast fusion-mediated
transfection (see Sambrook et al., Molecular Cloning A Laboratory
Manual, 2nd edition, Cold Spring Harbor Laboratory Press,
1989).
[0113] An agent that modulates Mgat5 activity, the amount of Mgat5
modified glycans, or the amount of binding of MgatV modified
glycans and lectins (e.g. galectins) may comprise a complex of a
lectin (e.g. galectin) associated with a Mgat5 modified glycan
and/or a polylactosamine modified glycan, or a lectin-Mgat5
modified glycan lattice; a peptide derived from the binding domain
of a lectin (e.g. galectin) that interacts with Mgat5 modified
glycan or polylactosamine modified glycan; and/or an
oligosaccharide derived from the Mgat5 modified glycan that
interacts with a lectin (e.g. galectin).
[0114] The reagents suitable for applying the methods of the
invention to evaluate substances and compounds that modulate
receptor clustering, more particularly T cell receptor clustering,
may be packaged into convenient kits providing the necessary
materials packaged into suitable containers. The kits may also
include suitable supports useful in performing the methods of the
invention.
Regulating Receptor Clustering
[0115] The invention provides a method for regulating receptor
clustering on cell surfaces comprising altering glycans on the cell
surface associated with receptor clustering, in particular altering
receptor glycosylation. Preferably the receptors are those that
comprise Mgat5 modified glycans or polylactosamine modified
glycans. Examples of receptors include receptors that stimulate
immune reactions (e.g. T cell receptors, Ig receptors, B cell
receptors, NK receptors), the HER family of transmembrane receptor
tyrosine kinases [e.g. epidermal growth factor (EGF) receptor also
known as HER1 or Erb1, HER2 (neu, Erb2), HER3 (Erb3), and HER4
(Erb4)], cadherin receptors (e.g. E-cadherin and N-cadherin),
interleukin (IL) receptors including IL-2 receptor, TNF.gamma.
receptor, and integrins.
[0116] Glycosylation may be altered by modulating one or more
glycosyltransferase enzyme involved in the synthesis of glycans
involved in receptor clustering, in particular N-glycans and
N-glycan intermediates (e.g. Mgat5 modified glycans or
polylactosamine modified glycans). Altering glycosylation may
involve increasing or decreasing Mgat5 modified glycans or
polylactosamine modified glycans associated with receptor
clustering. In a preferred embodiment, an enzyme involved in the
synthesis of the glycans is modulated (e.g. Mgat5).
[0117] In accordance with a particular aspect, the present
invention relates to a method for regulating receptor clustering on
cell surfaces, in particular ligand-dependent receptor clustering,
more particularly T cell receptor clustering comprising modulating
Mgat5 activity, the amount of Mgat5 modified glycans,
polylactosamine modified glycans, or lectin-Mgat5 modified glycan
lattice, or the amount of binding or interaction of one or more of
MgatV modified glycans or polylactosamine modified glycans and
lectins that interact with the glycans (e.g. galectins).
[0118] Receptor clustering or oligomerization, in particular
ligand-dependent receptor clustering, may be reduced or inhibited
by increasing the amount or levels of Mgat5 modified glycans,
polylactosamine modified glycans, and/or lectin-Mgat5 modified
glycan lattice, increasing the activity or amount of one or more
glycosyltransferase enzyme, or enhancing the interaction between
glycans involved in receptor clustering and substances that bind to
the glycans that regulate receptor clustering (e.g. lectins).
[0119] Receptor clustering, in particular ligand-dependent receptor
clustering, may be enhanced or increased and glycosylation of the
receptor may be altered by decreasing the amount or levels of Mgat5
modified glycans, polylactosamine modified glycans, and/or
lectin-Mgat5 modified glycan lattice, decreasing the activity or
amount of one or more glycosyltransferase enzyme, or inhibiting the
interaction between glycans involved in receptor clustering and
substances that bind to the glycans that regulate receptor
clustering.
[0120] In an aspect of the invention a method is provided for
lowering T cell activation threshold to agonists comprising
decreasing Mgat5 modified glycans, polylactosamine modified
glycans, or galectin-Mgat5 modified glycan lattice on the surface
of the cells, or dissociating galectin from such glycans or lattice
thereby lowering the T cell activation threshold. Mgat5 modified
glycans, polylactosamine modified glycans, or galectin-Mgat5
modified glycan lattice on the surface of the cells may be
decreased by inhibiting a glycosyltransferase such as Mgat5.
[0121] In another aspect, a method is provided for restricting T
cell receptor recruitment in response to an agonist or increasing T
cell activation threshold comprising increasing Mgat5 modified
glycans, polylactosamine modified glycans, or galectin-Mgat5
modified glycan lattice on the surface of the T cells, or enhancing
the interaction between one or more components of a galectin-Mgat5
modified glycan lattice (e.g. a galectin and glycans of the
lattice) thereby increasing the T cell activation threshold. The
amount of glycans or lattice on the surface of the cells may be
increased by increasing the levels or activity of one or more
glycosyltransferase enzyme (e.g. Mgat5).
[0122] In accordance with another aspect of the invention, a method
is provided for treating or preventing a condition associated with
decreased or increased receptor clustering or a receptor clustering
defect in a subject comprising altering glycans associated with or
involved in receptor clustering. Glycans can be altered or modified
by modulating a glycosyltransferase enzyme involved in the
synthesis of the glycans.
[0123] In accordance with a particular aspect of the invention, a
method is provided for treating or preventing a condition
associated with decreased or increased receptor clustering, more
particularly T cell receptor clustering, comprising modulating
Mgat5 activity, the amount of Mgat5 modified glycans,
polylactosamine modified glycans, and/or lectin-Mgat5 modified
glycan lattice, and/or the amount of binding or interaction of one
or more MgatV modified glycans, polylactosamine modified glycans
and lectins e.g. galectins.
[0124] A receptor clustering defect may be involved in conditions
such as autoimmune diseases or proliferative disorders such as
cancer.
[0125] A condition associated with increased T cell receptor
clustering may include a T cell mediated autoimmune disease such as
insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoid
arthritis, myasthenia gravis, systemic lupus erythematosus,
autoimmune hemolytic anemia, glomerulonephritis, enhanced delayed
type hypersensitivity, allergic conditions, hypersensitivity, and
autoimmune encephalomyelitis. Conversely, T cell recognition of
cancers and immune therapy of cancer is limited by weak stimulation
of T cells by tumor cells. The present invention may also be used
to treat cancers susceptible to immune modulation.
[0126] In an aspect the invention contemplates a method for
treating or preventing an autoimmune disease in a subject
comprising reducing T cell receptor clustering in the subject. T
cell receptor clustering is reduced by increasing the amount of
Mgat5 modified glycans, polylactosamine modified glycans, and/or
lectin-Mgat5 modified glycan lattice on the surface of T cells of
the subject. In an embodiment, the method comprises up regulating
or increasing the amount of Mgat5.
[0127] In an aspect the invention contemplates a method for
treating or preventing cancer in a subject comprising increasing T
cell receptor clustering in the subject. T cell receptor clustering
is increased by decreasing the amount of Mgat5 modified glycans,
polylactosamine modified glycans, and/or lectin-Mgat5 modified
glycan lattice on the surface of T cells of the subject. In an
embodiment, the method comprises down regulating or decreasing the
amount of Mgat5.
[0128] In an embodiment of the invention, a method is provided for
treating or preventing a condition associated with a growth factor
receptor, in particular epidermal growth factor receptor,
comprising regulating clustering or oligomerization (e.g.
dimerization) of the growth factor receptor by altering
glycosylation of the receptor, modulating Mgat5 activity, the
amount of MgatV modified glycans, polylactosamine modified glycans,
and/or the binding of MgatV modified glycans or polylactosamine
modified glycans and lectins for the glycans. Inhibition of growth
factor receptor clustering may be useful in treating conditions
involving aberrant growth factors including but not limited to
cancers such as solid human cancers, NSCL, breast cancer, head and
neck cancer, gastric cancer, prostate cancer, bladder cancer,
ovarian cancer, colorectal cancer, glioblastomas, and renal cell
carcinoma.
[0129] One or more agents may be used to regulate receptor
clustering. In particular, one or more agents may be used to
modulate glycosyltransferase activity, more particularly Mgat5
activity, the amount of Mgat5 modified glycans or polylactosamine
glycans, the amount of binding of MgatV modified glycans or
polylactosamine modified glycans and lectins that interact with the
glycans (e.g. galectins), or the amount of lectin-Mgat5 modified
glycan lattice.
[0130] Agents that modulate glycosyltransferase activity, more
particularly Mgat5 activity, include known inhibitors or enhancers
of glycosyltransferases, compounds or substances identified using
the methods described herein, nucleic acid encoding the
glycosyltransferases, and antisense sequences of the nucleic acid
sequence encoding the glycosyltransferases. Examples of
glycosyltransferase inhibitors and enhancers are illustrated herein
for Mgat5.
[0131] By way of example, known inhibitors of Mgat5 include an
analog of the acceptor substrate for Mgat5, .beta.GlcNAc
(1,2).alpha.Man(1,6).beta.ManOR, where the reactive 6'OH group has
been removed (Palcic, M. M. et al., J. Biol. Chem. 265 (12)
6759-6769). Inhibitors of enzymes earlier on in the Golgi
oligosaccharide processing pathway may also be used to inhibit
Mgat5 activity. Examples of inhibitors of other enzymes in the
Golgi oligosaccharide processing pathway include mannosidase
inhibitors such as swainsonine, 1,5-dideoxy-1,5-imino-5-mannitol
and 1,4-dideoxy-1,4-imino-D-mannitol.
[0132] Recombinant molecules containing the nucleic acid sequence
Mgat5 in an antisense orientation may be used to inhibit Mgat5
activity. The nucleic acid sequence shown in Saito, H. et al.
Biochem. Biophys. Res. Comm. 198; 318-327, 1994, or parts thereof,
may be inverted relative to their normal presentation for
transcription to produce antisense nucleic acid molecules. The
antisense nucleic acid molecules may be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known
in the art. The antisense nucleic acid molecules or parts thereof,
may be chemically synthesized using naturally occurring nucleotides
or variously modified nucleotides designed to increase the
biological stability of the molecules or to increase the physical
stability of the duplex formed with mRNA or the native gene e.g.
phosphorothioate derivatives and acridine substituted nucleotides.
The antisense sequences may be produced biologically using an
expression vector introduced into cells in the form of a
recombinant plasmid, phagemid or attenuated virus in which
antisense sequences are produced under the control of a high
efficiency regulatory region, the activity of which may be
determined by the cell type into which the vector is
introduced.
[0133] The amount of Mgat5 modified glycans may be increased in
cells by administering Mgat5, a nucleic acid molecule encoding
Mgat5, an agent that stimulates Mgat5, or a complex of the
invention. Increased cell surface Mgat5 modified glycans may
enhance the lectin-Mgat5 modified glycan lattice (e.g.
galectin-Mgat5 modified glycan lattice) at the cell surface so as
to restrict receptor clustering (e.g. T cell receptor clustering).
The amount of polylactosoamine modified glycans may be increased in
cells by administering one or more enzyme necessary for the
production of the glycans, a nucleic acid molecule encoding the
enzyme, an agent that stimulates the enzyme, or a complex of the
invention. Increased cell surface polylactosamine modified glycans
may enhance the lectin-Mgat5 modified glycan lattice (e.g.
galectin-Mgat5 modified glycan lattice) at the cell surface so as
to restrict receptor clustering (e.g. T cell receptor clustering).
These approaches may be useful in the prevention and treatment of T
cell mediated autoimmune diseases.
[0134] Agents, compounds, and substances described herein or
identified using a method of the invention may be formulated into
pharmaceutical compositions for administration to subjects in a
biologically compatible form suitable for administration in vivo.
By "biologically compatible form suitable for administration in
vivo" is meant a form of the substance to be administered in which
any toxic effects are outweighed by the therapeutic effects. The
substances may be administered to living organisms including
humans, and animals. Administration of a therapeutically active
amount of the pharmaceutical compositions of the present invention
is defined as an amount effective, at dosages and for periods of
time necessary to achieve the desired result. For example, a
therapeutically active amount of a substance may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of antibody to elicit a desired
response in the individual. Dosage regima may be adjusted to
provide the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0135] The active substance may be administered in a convenient
manner such as by injection (subcutaneous, intravenous, etc.), oral
administration, inhalation, transdermal application, or rectal
administration. Depending on the route of administration, the
active substance may be coated in a material to protect the
compound from the action of enzymes, acids and other natural
conditions that may inactivate the compound.
[0136] The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985). On this basis, the compositions include,
albeit not exclusively, solutions of the substances or compounds in
association with one or more pharmaceutically acceptable vehicles
or diluents, and contained in buffered solutions with a suitable pH
and iso-osmotic with the physiological fluids.
[0137] The activity of a pharmaceutical composition, an agent,
compound, or substance described herein or identified using a
method described herein may be confirmed in animal experimental
model systems.
[0138] In accordance with an aspect of the invention there is
provided a method of, and products for, diagnosing and monitoring
conditions characterized by an abnormality in receptor clustering
comprising assaying for differential glycosylation of the receptor.
Differential glycosylation may be assayed by determining the
presence of Mgat5 modified glyeans, polylactosamine modified
glycans, lectin-Mgat5 modified glycan lattice, or an alteration or
change in such glycans or lattice, compared to a control.
[0139] In an embodiment, a method of, and products for, diagnosing
and monitoring conditions characterized by an abnormality or defect
of receptor clustering involving the interaction of a galectin and
Mgat5 modified glycan or polylactosamine modified glycan is
provided comprising determining the presence of one or more of a
complex of the invention, a Mgat5 modified glycan, a
polylactosamine modified glycan, a galectin-Mgat5 modified glycan
lattice, or one or more of an altered Mgat5 modified glycan,
polylactosamine modified glycan, or a galectin-Mgat5 modified
glycan lattice.
[0140] Nucleic acid molecules encoding MgatV, an Mgat5 polypeptide,
and antibodies specific for Mgat5, complexes, lattice,
oligosaccharides, or peptides of the invention may be used in the
prognostic and diagnostic evaluation of conditions associated with
increased or decreased receptor clustering more particularly T cell
receptor clustering, and the identification of subjects with a
predisposition to such conditions. In an embodiment, the nucleic
acid molecules, Mgat5, and antibodies may be used in the diagnosis
and staging of T cell mediated autoimmune diseases.
[0141] The following non-limiting example is illustrative of the
present invention.
EXAMPLE
Methods
[0142] Delayed-type hypersensitivity (DTH) skin reaction: To induce
delayed-type hypersensitivity, 100 .mu.l of 5% (w/v)
4-ethoxymethilene-2-phenyl-2-oxazolin-5-one (oxazolone) (Sigma) in
ethanol/acetone (3:1, v/v) was injected epicutaneously to the
shaved backs of the 129/sv mice. Four days after sensitization, 25
.mu.l of 1% (w/v) oxazolone was applied on each side of the right
ear, and the left ear received 25 .mu.l of olive oil/acetone on
each side. Ear swelling was measured with a micrometer at 24 h
intervals for the next 5 days, and swelling was reported as the
difference between the ear thickness of the right minus the left
ears. [0143] EAE model: Mice (129/sv) 8-12 weeks of age were
injected subcutaneously with 100 .mu.l of rabbit MBP (Sigma)
emulsified 1:1 with complete Freunds adjuvant at three different
total doses (25, 100 and 500 .mu.g/mouse). Mice were observed from
day 5 to day 50, and observations were done blinded with respect to
the genotype until day 36. For lower doses of 25 and 50
.mu.g/mouse, half the total was injected on day 0 in the right
flank and the other half on day 7 in the left flank. The high dose
of 500 .mu.g/mouse was injected all on day 0 at the base of the
tail, and 500 ng of pertussis toxin was injected via the tail vein
on day 0 and day 2. [0144] T cell proliferation: Naive T-cells were
purified from spleens of 8-12 week old mice by negative selection
using CD3.sup.+ T cell purification columns (R&D laboratories)
or by panning on plates pre-coated with anti-CD19 antibody
(Pharmingen). T cell proliferation was measured by culturing cells
for 48 h in RPMI, 10% FCS, 10.sup.-5 M 2-mercaptoethanol in the
presence of one or more of the following soluble antibodies:
hamster anti-mouse CD3.epsilon. (clone 2C11; Cedarlane), hamster
anti-mouse TCR.alpha./.beta. (clone H59.72; Pharmingen) or 0.5
.mu.g/ml anti-mouse CD-28 (Pharmingen). PMA at 10 ng/ml and
ionomycin at 0.5 .mu.g/ml were also used to stimulate cells. Two
.mu.Ci of .sup.3H-thymidine were added for the last 20 h of
incubation, and cells were harvested on fiberglass filters and
radioactivity was measured in a .beta.-counter. [0145] TCR
clustering: Six-micron polystyrene beads (Polysciences) in PBS were
coated with hamster anti-mouse CD3.epsilon. antibody (Clone 2C11;
Cederlane) at 2 .mu.g/ml antibody followed by coating with 200
.mu.g/ml bovine serum albumin (BSA). To measure TCR clustering,
5.times.10.sup.4 T cells were incubated with 2.5.times.10.sup.5
anti-CD3.epsilon. antibody-coated beads in 100 .mu.l RPMI 1640+10%
FCS at 37.degree. C. for 10 minutes placed on poly-L-lysine coated
cover slips. The cells were fixed with 10% formalin, stained with 2
.mu.g/ml fluorescein isothiocyanate (FITC) labeled
anti-TCR.alpha./.beta. antibody (Pharmigen), solubilized with 0.2%
Triton.RTM. X-100 reagent, labelled with rhodamine-phalloidin and
Hoechst, and then visualized by deconvolution microscopy. For
disaccharide competition, wild type T cells were incubated for 20
min with 0, 0.01, 0.03, 0.09, 0.27, 0.8, and 2.4 mM of disaccharide
prior to exposure to anti-CD3.epsilon. antibody beads. To measure
TCR internalization, purified splenic T cells stimulated with
either 0.1 .mu.g/ml.sup.-1 anti-CD3 antibody or with 10 ng/ml PMA
for varying lengths of time were harvested and stained with
FITC-anti-TCR.alpha./.beta.. PMA concentrations were not limiting
as 10, 50 and 100 ng/ml produced similar internalization and cell
activation results. To measure actin reorganization, purified
splenic T cells, stimulated with 0.1 .mu.g/ml.sup.-1 anti-CD3 for
varying lengths of time, were fixed with 4% paraformaldehyde for 10
minutes, washed with PBS and stained with rhodamine-phalloidin and
mean fluorescence intensity (MFI) was determined by FACS. [0146]
TCR signaling: T cells (1.times.10.sup.6) and anti-CD3.epsilon.
antibody coated beads (5.times.10.sup.6 at 0.4 .mu.g/ml antibody)
in 100 .mu.l RPMI 1640 were pelleted, incubated at 37.degree. C.
for various times, then solublized with ice cold 50 mM Tris pH 7.2,
300 mM NaCl, 0.5% Triton.RTM. X-100 reagent, protease inhibitor
cocktail (Boehringer Mannheim) and 2 mM orthovanadate. Zap-70 was
immunoprecipitated by incubating whole cell lysates with rabbit
polyclonal anti-Zap-70 agarose conjugate (Santa Cruz) overnight at
4.degree. C., followed by one wash with lysis buffer and 3 washes
with PBS. Western blotting were done with whole cell lysates or
immunoprecipitates separated on SDS-PAGE gels under reducing
conditions, transferred electrophoretically onto PVDF membranes and
immunoblotted with antibodies to Akt/PKB (NEB), phospho-Akt/PKB
(NEB), phosphotyrosine (clone 4G10, Upstate Biotechnology), Zap 70
(clone Zap-70-6F7, Zymed), TCR.alpha. (polyclonal, Santa cruz) and
rabbit anti-galectin-3 (Dr. A Raz, University of Michigan). Cell
surface proteins were biotinylated using sulfosuccinimidobiotin
(NHS-biotin) for 30 min, PBS pH 8.0. Cells were lysed and labeled
protein was captured on streptavidin-agarose beads. To cross-link
surface proteins on purified naive T cells, the homobifunctional
cross-linker dithio-bis(sulfosuccinimydylpropionate (DTSSP) was
used at 0.1 mg/ml with 10.sup.6 cell/ml in PBS pH 8.0 for 10 min at
20.degree. C. T cells were preincubated for 20 min with or without
2 mM lactose, and reacted with DTSSP in the presence of the same.
Aliquots of cell lysate were immunoprecipitated with rabbit
anti-galectin-3 antibody or non-immune rabbit serum (NS), separated
on reducing-SDS-PAGE and Western blotted for CD3.epsilon. and
TCR.alpha. chain. The band above CD3.epsilon. is cross-reactivity
of secondary antibody with light-chain.
[0147] To measure Ca.sup.++ mobilization, purified T cells were
loaded with 10 .mu.M AM ester of Fluo-3 (Molecular Probes) washed
and stimulated with 10 .mu.g/ml of anti-CD3.epsilon. antibody at
37.degree. C. Emission at 525 nm was taken using a
spectrofluorimeter with excitation at 488 nm. Data is plotted as a
fraction of the Ca.sup.++ mobilized by addition of 2
.mu.g/ml.sup.-1 of ionomycin. LacZ activity in Mgat5.sup.-/- T
cells was detected by loading cells with
flourescin-di-.beta.-D-galactopyranoside (FDG) (Molecular Probes)
at 10.degree. C., and allowing the reaction to proceed for 30 min.
The reaction was stopped by the addition of 1 mM
phenyl-.beta.-thiogalactoside.
Results and Discussion
[0148] To explore the role of Mgat5 in T cell immunity,
Mgat5-deficient mice were examined for evidence of immune
dysfunction. Mgat5.sup.-/- mice are born healthy, and lack Mgat5
N-glycan products in all tissues examined (16). At 3 months of age,
peripheral white blood cells, erythrocyte and serum levels of IgM
and IgG were comparable in Mgat5.sup.-/-, Mgat5.sup.+/- and
Mgat5.sup.+/+ mice (data not shown). The CD4 and CD8 reactive T
cell populations in the spleen and thymus were also in the normal
range (FIG. 1B,C). At 12-20 months of age, an increased incidence
of leukocyte colonies in kidney and enlarged spleens were observed
in Mgat5.sup.-/- mice. Furthermore, 32% of the Mgat5.sup.-/- (6/19
mice) had macroscopic hematuria, mononuclear infiltrates and
extensive accumulation of fibrin within Bowman's space (crescents),
characteristic of proliferative glomerulonephritis (FIG. 1D). This
form of renal injury is often observed in autoimmune mediated
glomerulonephritis. Milder renal defects were observed in 68% of
the Mgat5.sup.-/- mice but not in the Mgat5.sup.+/- or
Mgat5.sup.+/+ mice (0/19).
[0149] To examine T cell responses in the mice, a type IV
delayed-type hypersensitivity (DTH) reaction was induced and tissue
swelling was measured. The protein-reactive hapten oxazolone was
applied topically to the backs of the mice, then again 4 days later
to the right ear. Ear swelling in Mgat5.sup.+/+ mice peaked 24
hours post application, and swelling was completely gone by day 5.
Ear swelling in Mgat5.sup.-/- mice attained a higher maximum
between 48 and 72 h, and persisted for a longer time (FIG. 1E). To
study T cell dependent autoimmunity in vivo (17), experimental
autoimmune encephalomyelitis (EAE) was induced by immunizing mice
with myelin basic protein (MBP) at 3 doses: 25, 100 and 500
.mu.g/mouse. At the lowest dose of MBP, 25 .mu.g/mouse, the
incidence of EAE was significantly greater in Mgat5 deficient mice.
Furthermore, 25 and 100 .mu.g/mouse doses of MBP produced more
severe EAE in Mgat5.sup.-/- mice compared to wild type littermates,
characterized by an earlier onset, greater motor weakness and more
days with disease (Table 1). Myelin iniections of 500 .mu.g/mouse
induced disease in all mice with greater peak scores and no
significant differences in disease incidence or severity between
genotypes. These results indicate that mice lacking Mgat5-modified
glycans are more susceptible to DTH and EAE autoimmune disease.
[0150] In vitro, splenic T cells from Magt5-/- mice
hyperproliferated in response to anti-TCR.alpha./.beta. antibody
(FIG. 2A). To examine this hypersensitivity in more detail,
purified ex vivo T cells were cultured at low density and
stimulated with increasing concentrations of soluble
anti-CD3.epsilon. antibody in the presence or absence of anti-CD28
antibody (FIG. 2B). Both the Mgat5 deficiency and CD28 engagement
reduced the requirements for TCR agonist as indicated by D.sub.50
values and were additive when combined (FIG. 2C). Furthermore, the
apparent Hill coefficient (n.sub.H), a measure of synchrony in the
responding cell population, was increased by both the Mgat5
deficiency and by CD28 engagement. Therefore, the stimulatory
effects of the Mgat5 mutation and CD28 co-receptor engagement were
additive and similar in potency.
[0151] Alterations in cell surface TCR complex levels and
intracellular signaling potential of T cells were examined and
ruled-out as possible causes of the Mgat5.sup.-/- hypersensitivity.
The Mgat5 deficiency did not significantly alter cell surface
expression of CD3, CD4, CD8, TCR.alpha./.beta., CD28 or CTLA-4
glycoproteins in resting T cells (FIG. 1B,C and data not shown).
Intracellular signaling potential in Mgat5.sup.-/- T cells is
normal, as treatment with the phorbol ester PMA and the Ca.sup.++
ionophore ionomycin stimulated T cells equally well from mice of
both genotypes (FIG. 2D).
[0152] The relationship between cell surface Mgat5-modified glycans
and T cell activation was examined. Leukoagglutinin (L-PHA) is a
tetravalent plant lectin and commonly used T cell mitogen that
binds specifically to Mgat5-modified glycans. Mgat5.sup.-/- T cells
were completely unresponsive to L-PHA, confirming that
Mgat5-modified glycans are required for stimulation by this lectin
(FIG. 2E). L-PHA reactive N-glycans are also present on B cells,
but L-PHA is not a B cell mitogen. Furthermore, B cell responses to
anti-IgM antibody, LPS and IL-4 plus anti-CD40 antibody were
similar for cells from Mgat5.sup.-/- and Mgat5.sup.+/+ mice (FIG.
2F and data not shown). In T cells, L-PHA induces signaling common
to TCR engagement, including phosphorylation of CD3.zeta.,
Ca.sup.++ mobilization, PKC-.gamma. and Ras/mitogen-activated
protein kinase (Mapk) activation (18; 19). The TCR.alpha./.beta.
chains have 7 N-glycans in total, and some are branched
complex-type structures with L-PHA reactivity (20; 21). These data
indicate that Mgat5-modified glycans are present on glycoproteins
of the TCR complex and required for L-PHA mitogenesis.
[0153] When bound to major histocompatibility complex
(MHC)/peptide, TCRs cluster with an inherent affinity greater than
unligated TCR and the stability of these clusters is critical for
intracellular signaling (22). However, the density of TCRs measured
at the site of T cell-APC (antigen-presenting cell) contact is only
marginally increased relative to the remaining cell surface,
leaving the majority of the TCRs unengaged by MHC/peptide (4). It
is possible that ligand induced TCR clustering in the plane of the
membrane may be increased in the absence of Mgat5-modified glycans,
thus lowering Mgat5.sup.-/- T-cell activation thresholds. To
visualize TCR reorganization in response to an antigen-presenting
surface, polystyrene beads were coated with anti-CD3.epsilon.
antibody and incubated with purified ex vivo T cells. After 10
minutes of contact, TCRs in Mgat5.sup.-/- cells was markedly more
concentrated at the bead surface compared to Mgat5.sup.+/+ cells
(FIG. 3A,B). TCRs on wild type cells could not be induced to
cluster to the same extent as Mgat5.sup.-/- cells even with longer
incubations (20 min) or using anti-CD3.epsilon. plus anti-CD28
coated beads (data not shown). Actin microfilaments were more
concentrated at the bead contact site in Mgat5.sup.-/- cells, and
overlapped more extensively with TCR in the merged images compared
to Mgat5.sup.+/+ T cells (FIG. 3A,B). TCRs are internalized
following productive TCR clustering (1), and this was significantly
greater in Mgat5.sup.-/- compared to Mgat5.sup.+/+ cells (FIG. 3C,
solid lines). Intracellular signaling mediated by PMA treatment
induces TCR internalization but at similar rates in Mgat5.sup.-/-
and Mgat5.sup.+/+ cells (FIG. 3C, dotted lines). Microfilament
re-organization was more rapid in Mgat5 deficient T cells following
soluble anti-CD3.epsilon. antibody stimulation (FIG. 3D).
Akt/protein kinase B (PKB) phosphorylation is dependent upon
phosphoinositide 3-OH kinase activity, which stimulates Rac/CDC42
GTPases and actin filament re-organization (23). Phosphorylated
Akt/PKB displayed a greater fold increase in Mgat5.sup.-/- compared
to Mgat5.sup.+/+ T cells (FIG. 3D). Mobilization of intracellular
Ca.sup.2+ following stimulation with soluble anti-CD3.epsilon.
antibody was enhanced in the absence of Mgat5-modified glycans
(FIG. 3E). Tyrosine phosphorylation of multiple proteins was
increased and persisted longer in Mgat5.sup.-/- T cells exposed to
anti-CD3.epsilon. antibody coated beads. (FIG. 3F).
Immunoprecipitation of Zap70 revealed increased phosphorylation in
Mgat5.sup.-/- cells 1 to 5 min following stimulation. Zap70 kinase
binds to dual phosphorylated immunoreceptor tyrosine-based
activation motif domains of CD3.zeta., and association of the
latter with Zap70 was increased in Mgat5.sup.-/- compared to
Mgat5.sup.+/+ T cells (FIG. 3G). In summary, the Mgat5 deficiency
enhanced ligand-dependent TCR aggregation, and consequently, signal
transduction and microfilament re-organization.
[0154] The larger size of Mgat5-modified glycans may limit the
geometry and spacing of TCR clusters in the plane of the membrane
(24). Alternatively, Mgat5-modified glycans may bind cell surface
galectins which restrict TCR mobility, thus antigen-induced TCR
clustering. The galectins are a widely expressed family of
mammalian lectins defined as N-acetyllactosamine-binding proteins.
The poly N-acetyllactosamine sequences preferentially added to
Mgat5-modified glycans (6), enhanced the affinity for galectin
binding (FIG. 1A). Galectins bind to lactosamine and lactose with
dissociation constants in the 10.sup.-4 M range (7; 8), an affinity
comparable to MHC/peptide-induced oligomerization of TCRs in
solution (22). Therefore, the avidity of a multivalent
galectin-Mgat5 glycoprotein lattice at the cell surface may be
sufficient to restrict TCR clustering. To probe for the presence of
galectin-glycoprotein interactions, wild type ex vivo T cells were
pre-incubated with various disaccharides for 20 min prior to a 10
min. stimulation with anti-CD3.epsilon. antibody coated beads.
Pre-incubation with lactose increased TCR clustering at the bead
interface and reduced TCR density elsewhere on the cells (FIG. 4C),
which is similar to the behavior of untreated Mgat5.sup.-/- T cells
(FIG. 3A,B). TCR clustering was not altered by pre-incubation with
the control disaccharide sucrose (FIG. 4B). Lactosamine and lactose
both enhanced protein phosphorylation induced by anti-CD3.epsilon.
antibody coated beads but sucrose and maltose had no effect (FIG.
4D and data not shown). Lactose did not enhance signaling in
Mgat5.sup.-/- T cells (data not shown).
[0155] Galectin-3 was detected on the surface of naive T-cells by
labeling with NHS-biotin, capture with streptavidin beads and
Western blotting with anti-galectin-3 antibodies (FIG. 4E).
Chemical cross-linking of the cell surface to stabilize complexes,
followed by western blotting of galectin-3 immunoprecipitates
demonstrated that galectin-3 is associated with TCR complex
proteins. This interaction was disrupted by either Mgat5 deficiency
or by incubating wild type T-cells with 2 mM lactose (FIG. 4E).
Taken together, the data demonstrates that a multivalent cell
surface galectin-glycoprotein lattice limits TCR clustering in
response to agonist, the avidity of which is dependent upon
Mgat5-modified glycans (FIG. 4F). The full complement of
glycoproteins and lectins present in the T cell lattice remain to
be defined but at a minimum includes galectin-3 and the TCR
complex. Others have shown that exogenously added galectin-1 binds
CD2, CD3, CD4, CD7, CD43 and CD45 and these proteins may also
participate in the lattice (25). Indeed, exogenous galectin-1
modulates T cell activation in vitro (9; 25), antagonizes TCR
signaling (26), and when injected into mice, it suppresses the
pathology of EAE (27).
[0156] The gene replacement vector used to produce the
Mgat5-deficient mice contained the reporter gene LacZ replacing the
first exon, which was expressed with the same tissue-specificity as
Mgat5 transcript (16). Both LacZ expression and cell-surface
Mgat5-modified glycans in Mgat5.sup.+/- T cells, respectively,
increased 48 h after stimulation demonstrating regulation of Mgat5
by transcriptional means (FIG. 4G,H). This suggests that Mgat5
enzyme activity and glycan production are limiting in resting T
cells, and with stimulation, increases in Mgat5-modified glycans
and galectins may dampen TCR sensitivity to antigen. Negative
feedback by Mgat5-modified glycans on TCR sensitivity is delayed as
it requires Mgat5 gene expression, which is dependent on T cell
activation status, and only indirectly on antigen concentrations.
This form of slow-negative regulation governed by steady-state
activity of the system is a key feature of robust and adaptive
biochemical pathways (28) and Mgat5-modified glycans may contribute
this feature to T cell regulation.
[0157] Viola et al have estimated that sustained clustering of 8000
TCRs is required for T cell activation, but other molecular
interactions clearly alter this threshold. With CD28
co-stimulation, only .about.1500 TCRs are required (2).
Co-signaling through CD28 decreases the extent of TCR clustering
needed for activation predominantly by recruiting protein
kinase-enriched GM1 ganglioside rafts to the site of TCR
engagement, thereby amplifying signaling (3; 5). Here it is shown
that Mgat5 deficiency increases the number of TCRs recruited to the
antigen-presenting surface, thereby reducing the requirement for
CD28 co-receptor engagement. This may lead to T cell activation in
the absence of CD28 co-signaling, failure of anergy and loss of
immune tolerance. CD28.sup.-/- mice are resistant to induction of
EAE by low dose MBP, while Mgat5.sup.-/- are hypersensitive, but
both mutants develop clinical signs of EAE comparable to wild type
littermates with high doses of MBP (29). In this regard, CD28 and
Mgat5 appear to be opposing regulators of T cell activation
thresholds, and susceptibility to autoimmune disease. In summary,
Mgat5-dependent glycosylation limits agonist-induced TCR clustering
by sequestering receptors in a cell surface galectin -glycoprotein
lattice. However, the glycosylation deficiency in Mgat5.sup.-/-
mice affects other pathways and cells types that may also
contribute to the observed autoimmunity. Indeed, Mgat5-modified
glycans also reduce clusters of fibronectin receptors causing
accelerated focal adhesion turnover in fibroblasts and tumor cells;
a functionality that may affect leukocyte motility (16). Finally,
glycosylation of Notch receptor by Fringe, a fucose-specific
.beta.1,3GlcNAc-transferase provides another example of regulation
by differential receptor glycosylation (30). In a broad context,
the results described herein suggest a general mechanism for the
regulation of receptor clustering through differential
glycosylation and interaction with cell surface lectins.
TABLE-US-00001 TABLE 1 Clinical observations of autoimmune
encephalomyelitis (EAE) Incidence of Days with Groups (dose) EAE
Peak score Onset (days) disease Deaths Mgat5.sup.+/+ (25 .mu.g)
3/11 0.45 .+-. 0.24 24 .+-. 3.9 7.0 .+-. 3.9 0 Mgat5.sup.-/- (25
.mu.g) 9/11# 1.82 .+-. 0.39* 19.8 .+-. 3.3* 11.5 .+-. 3.0* 1
Mgat5.sup.+/+ (100 .mu.g) 10/10 1.6 .+-. 0.22 25 .+-. 2.2 18.5 .+-.
2.2 0 Mgat5.sup.-/- (100 .mu.g) 10/10 2.1 .+-. 0.34* 17.6 .+-. 2.9*
23.3 .+-. 3.5* 1 Mgat5.sup.+/+ (500 .mu.g) 12/12 3.0 .+-. 0.43 8.9
.+-. 1.2 27.9 .+-. 4.0 3 Mgat5.sup.-/- (500 .mu.g) 12/12 2.83 .+-.
0.38 9.3 .+-. 0.95 27.2 .+-. 2.9 2 Disease severity was scored on a
scale of 0-5; with 0, no illness; 1, limp tail; 2; limp tail and
hindlimb weakness; 3, hindlimb paralysis; 4, forelimb
weakness/paralysis and hindlimb paralysis; 5, moribund or death.
Mean .+-. SE of incidence, peak score, and days with disease were
calculated using the total number of mice injected per dose as the
denominator. The mean .+-. SE for day-of-onset was determined by
only using those mice that became sick. #Contingency test, P <
0.001; and *Mann Whitney test comparing genotypes for significant
differences at P < 0.05.
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[0188] The present invention is not to be limited in scope by the
specific embodiments described herein, since such embodiments are
intended as but single illustrations of one aspect of the invention
and any functionally equivalent embodiments are within the scope of
this invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0189] All publications, patents and patent applications referred
to herein are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety. All publications,
patents and patent applications mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the cell lines, vectors, methodologies etc. which are reported
therein which might be used in connection with the invention.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0190] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to the "antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
* * * * *