Download The predominant surface glycoproteins of thymocytes and

277
Bioscience Reports 2, 277-287 (1982)
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T h e p r e d o m i n a n t s u r f a c e g l y c o p r o t e i n s of
t h y m o c y t e s and l y m p h o c y t e s
Review
Alan F, WILLIAMS
MRC Cellular Immunology Unit, Sir William Dunn School of
Pathology, University of Oxford, Oxford OXI 3RE, U.K.
The surfaces of thymocytes and T and B lymphocytes
d i f f e r m a r k e d l y in t h e i r p r e d o m i n a n t glycoprotein
constituents. Amino acid sequence studies show that a
surprising number of t h e cell surface molecules have
homologies with immunoglobulins,
Analysis :of carbohydrate shows major differences between glycoproteins
on the same cell and between the same (or closely
related) glycoproteins on the different cell types,
Introduction
T h e r e are two main types of immunocompetent lymphocytes: B
lymphocytes, which differentiate in the bone marrow and give rise to
antibody-secreting cells, and T lymphocytes, which differentiate in t h e
thymus and are responsible for ceil-mediated immunity. The surface
molecules of both B and T lymphocytes have been intensively studied
and also those of immature T lymphocytes from the thymus (thymocytes).
Under the microscope mature B and T lymphocytes and most
thymocytes look very similar, with a large nuclear:cytoplasmic ratio,
few mitochondria, and an absence of endoplasmic reticulum.
These
features are consistent with the f a c t that these are non-dividing cells
w~th low levels of metabolism and biosynthetic activity.
tn the absence of antigen, lymphocytes have a migratory existence
during which they recirculate from blood to lymph.
This involves
s p e c i f i c r e c o g n i t i o n of s p e c i a l i z e d e n d o t h e l i u m in lymph nodes,
movement between endothelial cells, and migration into areas which
are different for B and T lymphocytes. The time of passage through
the nodes is much shorter for T lymphocytes than for B lymphocytes
(l).
If foreign antigen is encountered a complex set of recognition
events occurs.
B lymphocytes can bind antigen directly with their
immunoglobulin receptors but require interactions with macrophages
and T l y m p h o c y t e s to trigger cell division and differentiation tO
produce antibody-secreting cells and memory cells (2). T lymphocytes
a p p e a r to r e c o g n i z e antigen in association with histocompatibility
antigen presented on macrophages or dendritic cells.
The molecular
basis of this is not understood at all (3).
Given the above functions it seems reasonable to think that the
cell surface molecules of B and T lymphocytes will be mainly involved
with the recognition phenomena seen in recirculation and cell positioning, and antigen recognition.
For thymocytes, surface molecules
may be involved in cell positioning and in events leading to committment to a particular specificity of antigen recognition.
I would
01982
The Biochemical Society
278
WILLIAMS
expect that metabolite transport molecules or ion pumps would be
minor components of the membranes of these cells.
The situation
would presumably be different for the dividing precursor cells of the
early stages of lymphoid differentiation or the dividing cells resulting
from stimulation of mature lymphocytes with antigen. An example is
the transferrin receptor seen on dividing but not quiescent lymphocytes
(4).
In this article I will discuss the major surface glycoproteins of
thymocytes and of mature T and B lymphocytes with regard to three
m a i n a s p e c t s : (a) their patterns of expression and functions, (b)
homology of a number of molecules with immunoglobulins, and (c)
heterogeneity in their carbohydrate structures.
Methods
Monoclonat antibodies now provide the primary tools for identifying
celt surface proteins (reviewed in refs. 576)~ Purification has been
achieved using affinity chromatography with lectin or antibody columns
(5~7).
The assignment of a molecule as a major surface component
rests on a demonstration that it accounts for one of the major entities
seen in surface labelling studies and on determination of the number
of molecules per cell.
For example, on rat thymocytes the three
heavily glycosylated molecules given in Table 1 account for the three
main bands seen when sialic acid or galactose residues are labelled at
the cell surface with 3H borohydride (8).
Two of these molecules
(L-CA and Thy-1) account for the two main glycoproteins which bind
to lentil tectin and are identified by Coomassie Blue staining after
sodium dodecyI sulphate polacrylamide-gel electrophoresis (9).
LSGP
does not bind to lentil tectin, but most cell surface glycoproteins do,
as judged by the fact that most of the proteins tabelled with t25I by
t h e l a c t o p e r o x i d a s e method are bound by this lectin (g).
Taken
together these data suggest that there may be only three abundant
glycoproteins on thymocytes. Other molecules found at levels of about
10~ to 3 x 10~ molecules per cell have been described and include the
markers of T-lymphocyte subsets (5).
Patterns
o5 E x p r e s s i o n
Glycoproteins
and
Functions
o5 t h e
Major S u r f a c e
The known abundant constituents of lymphoid surfaces are given in
Table i, which shows data for rat lymphoid cells, The same patterns
seem to apply equally to mouse or human cells~ with the exception
that T h y - I antigen is found on lymphocytes as well as thymocytes in
the mouse and not on either in humans (reviewed in ref. tO) and that
the main human Class-II antigens are homologues of the rodent E type
( t 1).
A l l the m o l e c u l e s listed in Table 1 are glycoproteins or
multimers of protein and glycoprotein chains, and all but LSGP and
Thy-1 are known to traverse the lipid bilayer ( i 2 - 1 4 ) .
For LSGP
there are at present no data on membrane integration. Thy-1 has all
the properties of a molecule which binds to the lipid bilayer, but these
cannot be due to the polypeptide and there is evidence for a nonprotein hydrophobic 'tail' (10).
LYMPHOCYTE GLYCOPROTEINS
279
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280
WILLIAMS
Simple inspection of Table 1 shows that the different lymphoid cell
types are very different in their major cell surface constituents. The
only molecule in Table 1 which may be identical on any two cell types
is the Class-I histocompatibility antigen on T and B lymphocytes.
Thymocytes and T l y m p h o c y t e s both express the LSGP and all the
c e l l s express L-CAs.
However, the carbohydrate on these glycoproteins differs on the different cell types (see below). Presumably
the differences in surface molecules are related to the functions of
the cells.
However, for most glycoproteins this is difficult to think
a b o u t in a simple way because of their unpredictable expression
amongst various cell types, including non-lymphoid ceils (Table 1).
Of t h e m o l e c u l e s in Table i, f u n c t i o n s are known only for
immunoglobulin and histocompatibility antigens. Immunoglobulin is the
antigen receptor of B lymphocytes and is not known to be synthesized
by any other cell type (15). This molecule thus fits with the concept
of a uniquely expressed molecule mediating a particular function of a
differentiated cell. Histocompatibility antigens function as controlling
elements in T-lymphocyte responses (2,3).
T lymphocytes appear to
recognize foreign antigen only at the surface of a presenting cell and
t h i s s o m e h o w involves the s i m u l t a n e o u s r e c o g n i t i o n of h i s t o c o m p a t i b i l i t y antigens.
The part of the histocompatibility antigen
which i s recognized is that which shows polymorphism within the
species. Class-I histocompatibiiity antigens seem to be mainly involved
in recognition by cytotoxic T ceils and may be particularly relevant to
the killing of ceils infected with virus, while Ctass-II antigens are
recognized by helper T cells and are thus important in the initiation
of i m m u n e responses controlled by these ceils.
The wide tissue
d i s t r i b u t i o n of Class-I antigens thus seems to fit well with their
function, since most cell types might by infected by virus. HoweveG
the distribution of Class-II antigens at present seems bizarre, since
only macrophages and/or dendritic ceils are known to be involved in
the presentation of antigen to helper T cells.
The expression of LSGP and Thy-I antigen on various cell types is
as unusual as t h a t of Class-II antigens.
These antigens are all
d i s c r e t e l y e x p r e s s e d on some, but not all cell types without any
obvious functional correlation. This expression cannot be dismissed as
a serological a r t i f a c t since Class-II antigens (16), Thy-1 (17), and
LSGP (18) have all been characterized from different celt types and
the results show that the shared antigenicity reflects the expression of
a s i m i l a r molecule (probably identical in the polypeptide) on the
different cell types.
The phenomenon of unusual patterns of antigen
expression is clearly of major biological significance, since it applies
to other membrane molecules (5) as well as those shown in Table l.
The results of Structural studies on the major glycoproteins provide
some basic data for thinking about the functions of these molecules.
Homology with Immunoglobulins
Fig. l shows cell s u r f a c e m o l e c u l e s with known amino acid
sequences found on lymphoid cells.
These include histocompatibility
antigens, Thy-1 antigen, and immunoglobulins. These molecules have
all been studied for completely unrelated reasons yet they all turn out
to show sequence homology and are all likely to contain segments
homologous to the Ig-fold.
LYMPHOCYTE
GLYCOPROTEINS
281
Immunoglobulin-Related Molecules Found At Cell Surfaces
IgM
V-domains
MHC Antigens "~r-~
", ~,,o
ClassI
Class][ C/~"rj~<~l.>=~) C~,~
<,al "-'t
TTTT ITTTTTTTI TTTTTTTTTT'
,IJTTI
Cytoplasm
Fig. 1,
Tmmunoglobulin-related molecules found at
lymphoid cell surfaces.
Ig domains and their
homologous regions in the other molecules are
represented by circles.
Intra-chain disulphide
bonds
are shown by the ~ symbols and interchain
bonds in IgM by dashed lines. N-linked carbohydrate
structures are shown by ~ and n and c identify the
N-terminus and C-terminus of the polypeptides with
the exception of ~2-microglobulin (~2-m).
The
polymorphic
determinants of the major histocompatibility (MHC) antigens are found mainly in the
heavy chains of Class-I antigens and the 13 chains of
Class-ll antigens.
The diagrams are based on
references 10,19,34 (Thy-l); 42 (Class-I antigens);
ii,12~22-24~46 (Class-ll antigens); 14,47 (IgM).
The h e a v y and light chains of immunoglobulins can be subdivided
i n t o d o m a i n s of a b o u t 110 a m i n o acids which show s e q u e n c e and
structural homology (18).
This is i l l u s t r a t e d for an IgM m o l e c u l e in
Fig. i.
In all c a s e s (including V - d o m a i n s ) t h e s e domains a r e c h a r a c t e r i z e d by a c o n s e r v e d disulphide bond and a c h a r a c t e r i s t i c folding
p a t t e r n r e f e r r e d to as t h e I g - f o l d (18).
This consists of a n t i - p a r a l l e l
B - s t r a n d s f o r m i n g t w o B - s h e e t s w h i c h are held t o g e t h e r by the
disulphide bond and by hydrophobic i n t e r a c t i o n s b e t w e e n the sheets.
S e q u e n c e homologies b e t w e e n the I g - d o m a i n s a r e seen m o s t l y around
t h e c o n s e r v e d c y s t e i n y l residues and in s t r e t c h e s of s e q u e n c e which
f o r m the B-strands.
A p a t t e r n for V- or C - d o m a i n s can be distinguished by p a r t s of the s e q u e n c e which are c o n s e r v e d within but not
b e t w e e n t h e s e domains, and by h o m o l o g i e s in the middle of the domain
w h e r e V - d o m a i n s h a v e e x t r a s e q u e n c e c o m p a r e d with C - d o m a i n s . Thus
282
WILLIAMS
in looking for homologies with Ig the features to be noted are: the
c h a r a c t e r i s t i c disulphide bond, B - s h e e t s t r u c t u r e , and sequence
homologies.
B2-microglobulin (B2-m) and T h y - i antigen have all of these
features and both are homologous to a single immunoglobulin domain.
Thy-1 ~ t s best with V-domains (19) while B2-m is clearly like a
C-domain (20).
Parts of the Class-I heavy chain (2i) and Class-II a
(22) and B (23,24) chains also have homology with Ig domains in
terms of the conserved disulphide bond and sequence homology. In all
cases these are clearly like Ig C-domains rather than V-domains. Thus
all of the domain-like segments of histocompatibility antigens are
h o m o l o g o u s to C - d o m a i n s , and Thy-1 antigen is the only non-Ig
molecule so far identified which has a V-domain pattern.
Carbohydrate Heterogeneity
The nature oi the carbohydrate of lymphoid glycoproteins has been
studied by analysis of their composition and by measuring their binding
to l e c t i n s .
The r e s u l t s show that there are striking differences
b e t w e e n m o l e c u l e s on t h e same cell and between the same (or
related) molecules on different cells.
Cell surface glycoproteins can be labeIled with 3H at sialic acid or
galactose residues by reducing with 3H borohydride after oxidation with
p e r i o d i c acid, or g a l a c t o s e oxidase subsequent to neuraminidase
digestion to expose galactose residues (25). When this is done only a
few heavily labelled glycoproteins are seen, namely L-CA, LSGP, and
Thy-1 on thymocytes, L-CA and LSGP on T tymphocytes, and L-CA
only on B lymphocytes (8).
The thymocyte molecules have all been
purified (26) and their carbohydrate compositions show that the LSGP
molecule has very different carbohydrate structures from L-CA and
Thy-i (Table 2). The composition and size of the LSGP carbohydrate
structures suggest that one amino acid in five has attached to it an
O-linked structure of the type:
sialic acid
c~2-~ Gal 81-3~ GaINAc
ta2-6
sialic acid
Ser/Thr.
The molecule is thus highly acidic and is likely to have extended
c o n f o r m a t i o n , since t h e a t t a c h m e n t of such a large number of
carbohydrate structures might be expected to inhibit protein folding.
S t u d i e s on binding of glycoproteins to lectins have established
differences in their carbohydrates between cells.
For Thy-1 antigen,
differences were seen in binding to lentil lectin of the brain and
thymus forms of the antigen (17), and more recently peanut lectin
( P N L ) and soybean lectin (SBL) have been particularly useful in
a n a l y s i n g c a r b o h y d r a t e h e t e r o g e n e i t y b e t w e e n different types of
lymphoid cells (27).
PNL is specific for GalB1-3GalNAc disaccharide, which is the asialo
form of the O-linked structure given above.
PNL binds in large
amounts to thymocytes compared with T lymphocytes and has been
used to fractionate ceils from the thymus (28). The LSGP is clearly
LYMPHOCYTE
Table 2.
GLYCOPROTEINS
293
Carbohydrate compositions of the three major
rat-thymocyte glycoproteins
L-CA
Thy-i
LSGP
% Carbohydrate
25
32
60
Estimated structures per i00 amino acids
N-linked
1.5
3
nil
O-linked
few
nil
20
Residues per I00 amino acids
Fucose
0.9
1
0
Mannose
4.4
10.6
0
Galactose
3.6
5.5
22.3
Glucosamine
5.7
9.4
0
Galactosamine
1.1
0
20.1
Sialic acid
2.9
2.1
27.2
Data from refs. 17 and 26.
a candidate for interaction with PNL, and as expect ed the whole of
this glycoprotein from t h y m o c y t e s and T lymphocytes binds to a PNL
affinity column if terminal sialic acid is removed with neuraminidase.
With undigested cells part of the LSGP from t hym ocyt es binds to the
PNL column but no binding of the T - l y m p h o c y t e LSGP occurs (27).
This suggests that the differential binding of PNL is due simply to the
f a c t that t h y m o c y t e carbohydrate structures partially lack terminal
sialic acid while those of T lymphocytes are fully sialylated. Previous
studies in the mouse have shown that the same phenomenon occurs for
r h y - 1 antigen ( 2 9 ) .
T h y m o c y t e Thy-1 antigen partially lacks sialic
acid while T - l y m p h o c y t e Thy-l is fully sialylated. Thus it seems t hat
th e c a r b o h y d r a t e structures of t h y m o c y t e s may in general lack sialic
a c i d and t h a t in t h e d i f f e r e n t i a t i o n to mature lymphocytes the
glycosylation changes to give structures which have a full complement
of terminal sialic acid.
SBL is s p e c i f i c for terminal galactosamine and binds in large
amounts to 15 lymphocytes but virtually not at all to T lymphocytes.
In a f f i n i t y chromatography no glycoproteins from t h y m o c y t e s or T
lymphoctyes bound t o SBL, but a significant fraction of B-lymphocyte
L - C A was bound ( 2 7 ) .
A f t e r t h e r e m o v a l of sialic acid with
neuraminidase all of the B-lymphocyte L-CA bound to the SBL column
but still virtually no t h y m o c y t e or T - l y m p h o c y t e L-CA was bound. In
af f in ity chromatography with the PNL column, p a r t of the asialo L-CA
f r o m T l y m p h o c y t e s and B lymphocytes was bound but t h y m o c y t e
L-CA did not bind even in the asialo form. Taken t o g e t h e r these data
establish t hat L-CA differs in its c a r b o h y d r a t e st ruct ures on all t h r e e
28•
WILLIAMS
cell types, and this conclusion is supported by the report that L-CA
f r o m r a t s p l e e n ( f r o m B and T l y m p h o c y t e s ) expresses the 'i'
car b o h y d r at e antigenic determinant while the t h y m o c y t e form does not
(30).
L C - A shows e x t e n s i v e s e r o l o g i c a l c r o s s - r e a c t i o n between the
d i f f e r e n t cell types and it seems likely that the di fferent forms share
the same polypeptide with differing carbohydrate structures. However,
t h e r e are antibodies which distinguish B-lymphocyte L-CA(31,32) from
the other forms, and sequence studies will be essential to establish
whether the polypeptides are identical or whether the different L-CAs
are coded by a closely related but non-identical set of genes.
Discussion
A t the outset I suggested that the major molecules at the surface
of lymphocytes should be mainly involved in the recognition functions
which occur in antigen recognition or lymphocyte recirculation and
p o s i t i o n i n g in l y m p h o i d t i s s u e s .
For immunoglobulins and histocompatibility antigens this is so, with both sets of molecules being
involved in different ways in antigen recognition.
A relationship in
the functions of these molecules is now strongly supported by the
findings of homology in the sequences.
The functions of immunoglobulins are in part understood at the level of the individual domains
wh ile for h i s t o c o m p a t i b i l i t y antigens this is not so.
For histoc o m p a t i b i l i t y a n t i g e n s all t h a t is known is that somehow their
polymorphic parts must be recognized at the same time that foreign
antigen is recognized by T lymphocytes at a cell surface.
T h e f u n c t i o n s of immunoglobulins can be divided into antigen
r e c o g n i t i o n by V - d o m a i n s and mediation of e f f e c t o r functions by
C-domains.
Both sorts of domains have a similar folding pat t ern for
their polypeptide chain, and the different functions are mediated by
sequences which do not contribute to the structural stability of the
Ig-fold. These occur in the bends connecting the anti-parallel strands
and also possibly on the exposed surfaces of the B-strands (33). For
both V- and C-domains it could be argued that the essence of the
f u n c t i o n is t h a t t h e Ig-fold provides a stable platform on which
variations of sequence can occur and mediate di fferent recognition
functions.
This idea is clearly compatible with the function seen for
histocompatibility antigens.
Can the argument be extended to Thy-1
antigen?
The structural homologies would argue for this, and it has
b e e n s u g g e s t e d t h a t Thy-1 antigen functions as a ligand in cell
i n t e r a c t i o n s r e s p o n s i b l e for tissue formation (34).
A key point
regarding Thy-1 antigen is that it breaks the link between the Ig-fold
and immunological recognition functions. This is so since expression of
Thy-1 is conserved in different species in neuronal ceils and fibroblasts
but not on lymphoid cells (10).
If Thy-1 is involved in recognition
functions then the distribution of the antigen would suggest that it can
undertake a triggering role on more than one cell type.
The carbohydrate het er oge ne i t y of the surface glycoproteins is also
of interest in thinking about recognition phenomena.
The hypothesis
th at carbohydrates mediate cellular interactions by acting as ligands
for recognizers (glycosyl transferases or lectins) on other ceils has
long been discussed (35,36).
There is no direct evidence for this
LYMPHOCYTE GLYCOPROTEINS
285
hypothesis but clearly heterogeneity of the sort seen in the carbohydrates of lymphoid glycoproteins could be a basis for such interactions. It is established that the carbohydrates of enzymes (37) and
serum glycoproteins (3g) can play an informational role in the uptake
of t h e s e m o l e c u l e s by cells, and it is easy to imagine that the
carbohydrate of LSGP, L-CA, or Thy-I might be presented as ligands
for mediating cell interactions.
If this view is taken then one can
argue that the same antigen on different cell types could provide a
cell-specific ligand in that the carbohydrate may be cell-type-specific
(I7).
Alternatively one could argue that the carbohydrate is important
for structural reasons.
This seems to be the case for the N-linked
carbohydrate structure of IgG (39), and for Thy-l antigen correct
glycosylation is essential for the molecule to be expressed at the cell
surface (40). Structural aspects could include determining t h e charge
characteristics of the cell surface, and this may be the main role of
the LSGP.
P o s s i b l y t h e s u r f a c e carbohydrates will turn out to have both
i n f o r m a t i o n a l and structural roles.
Acknowledgements
t am grateful to Mrs. Chris Scott for assistance with the manuscript.
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