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Surface molecules and cell interactions
J. theor. Biol. (1982) 98,221-234
Surface Molecules and Cell Interactions ALAN F. WILLIAMS MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX 1 3RE England (Received
10 February
1982)
Many of the cell surface molecules of lymphocytes or their precursors are expressed in an unpredictable way on a limited set of other cell types. This often seems to involve expression on lymphoid and brain cells. The Thy-l antigen is in this category, being a major glycoprotein of murine neuronal cells, fibroblasts and thymocytes. Structural studies show that this molecule is homologous with immunoglobulin domains which are the structural sub-units of all immunoglobulin polypeptides. Thy-l is the size of one immunoglobulin domain and its sequence is most homologous with variable regions of immunoglobulins. It is suggested that Thy-l is one of a set of surface molecules concerned with triggering interactions between cells and that this is the primitive function of the immunoglobulin domain. Cell interactions could be medi-
ated by domain-like
structures and receptors for them in a way which
parallels the triggering of immunological effector reactions by the interaction of receptors with immunoglobulin constant regions. If this is so then the structure seen in the immunoglobulin domain would have evolved along with the evolution of cell organisation. The genes specifying the cell interaction molecules could then have provided the genetic material for the evolution of antibody and histocompatibility antigen at the time of vertebrate emergence.
Lymphocyte
Functions
and the Cell Surface
It seems highly likely that molecules on adjacent cell surfaces interact to influence the behaviour of cells. Amongst other things, such interactions
could provide start or stop signals for cell movement and essential triggers for division and differentiation of cells at various stages in their lineages. Cues for cell movement and differentiation are needed during embryogenesis (Bronner-Fraser & Cohen, 1980) and also in many aspects of the behaviour of cells in adult organisms. The lymphocyte is a good example of a cell type which is continuously involved in various interactions with other cells. In its resting state the 221
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lymphocyte recirculates from blood to lymph until being triggered to divide and differentiate by antigen. Lymphocyte recirculation involves specific recognition of specialised endothelium in lymph nodes and migration between the endothelial cells into positions which are different for B and T lymphocytes (Ford, Smith & Andrews, 1978). To mediate this behaviour there must be triggers for adhesion, movement and localisation of lymphocytes. The interaction of lymphocytes with antigen also involves cell-cell interactions since T lymphocytes appear to recognise antigen at the cell surface of macrophages (or another similar cell type) and B lymphocytes probably interact with macrophages and T lymphocytes in the process of being stimulated by antigen (basic text Hood, Weissman & Wood, 1978). These interactions with antigen and other cell types result in cell division and entry of lymphocytes into a new phase of differentiation. All these functions are of interest in their own right, but they may also provide a model for movement and differentiation of other cell types. As well as being functionally interesting there are technical reasons why lymphocytes are more suitable for cell surface studies than most other cell types. Lymphocytes undertake all their interactions without forming irreversible junctions with other cells and thus can be isolated without the use of proteolytic or other degradative enzymes. Large numbers of cells can be obtained (>lO”) and this allows the purification of membrane molecules for structural and functional studies. The molecules found at the surface of thymocytes and T and B lymphocytes are now being characterized. This is mostly being done by preparing monoclonal antibodies against mixtures of membrane molecules and then using the antibodies to identify the constituents of the membrane (Williams, Galfre & Milstein, 1977; Williams, 1980). As the molecules are first indentified with antibodies they are initially referred to as antigens and often by the arbitrary name of the monoclonal antibody used for their identification (Table 1). The studies on lymphoid membrane molecules are at an early stage but there have already been surprises. The unpredictable distribution of lymphocyte antigens on other cells presents one puzzle and unexpected structural homologies between surface antigens and immunoglobulins (Ig) present another. These may be reconciled in an hypothesis which suggests that there is a set of structures, including Ig-related molecules, which mediate cell interactions and can function in a similar way on various cell types. Others have speculated on related themes (Burnet, 1970; Bodmer, 1972; Gally & Edelman, 1972; Hood, Huang & Dreyer, 1977) but previous ideas will not be reviewed in this paper.
:
antigens
antigens:
antigen
antigen
:
lymphocytes
2 X 55 000 gp 2x25 ooogp
30 000 gp 28 000 gp
43 000 gp 12 000 p
nature mol. wt. (gp) protein (p)
21000p
gP
2 x lo4 thymocytes not known
gP
not known
47 000 gp
17500-187OOgp
95 000 gp
170000-220000gp
tetramer:
dimer:
dimer:
Molecular glycoprotein
3 x 10” thymocytes
1 S x lo4 thymocytes
2 x lo4 thymocytes
lo6 thymocytes
10’ thymocytes
7 x lo4 thymocytes
5 x 104B lymphocytes
1.5 x lo58
10’ lymphocytes
Sites per cell on cell named on’
only.
or
T lymphocytes.
Thymocytes, brain, follicular dendritic cells, endothelial cells. Thymocytes, T lymphocyte sub-set, macrophages Thymocytes, T lymphocyte sub-set, natural killer cCIIS. Thymocytes, all T lymphocytes.
Most haemopoietic cells with different carbohydrate on different cell types. Thymocytes, T lymphocytes, polymorphs, plasma cells, brain Thymocytes, neuronal cells, fibroblasts, connective tissue, myoblasts, immature B lymphocytes and haemopoietic stem cells.
B lymphocytes
Many cell types but not neuronal glial cells, also very low on most thymocytes. B lymphocytes, dendritic cells, activated macrophages, epithelial cells of various organs.
Expressed
t Data from references reviewed in Williams (1980); Brown et al. (1981); Campbell er al. (1981) plus additional data from Bjiirck, AkerstrGm % Berggird (1979); Thiele, Arndt, Stark & Wonigeit (1979); Barclay (1981a) and (19816) and Barclay, A. N., Dallman, M. & Ward, H., unpublished observations. D Antigen is present on the named cell types and not found on other cell types seen in tissue sections of lymphoid organs, kidney, brain. gut or liver.
Pta (Ly-2)
OX
MRC
19 antigen
OX 8 antigen
antigen
MRC
W3/25
Minor glycoproteins and antigens MRC OX 2 antigen
Leucocyte sialoglycoprotein (W3/13 antigen) Thy-l antigen
Leucocyte-common
Major glycoproreins
Immunoglobulin:
Class II
Major histocompafibility Class I
Cell surface
1
Some cell surface antigens of rat lymphocytes?
TABLE
z W
224
A.
The Tissue Distribution
F.
WILLIAMS
of Lymphocyte
Cell Surface Molecules
The pattern of distribution on other cell types of molecules (antigens) found at the lymphocyte surface is surprising. Arguing on a simple-minded basis it might be expected that cell-surface molecules would either be widely distributed on most cells or tightly restricted to one or a few functionally related cell types. Molecules involved in basic metabolic functions would be widely distributed and those concerned with the specialised functions of cells restricted. For lymphocytes most surface molecules do not fit into either of these categories but rather are found on various but not all cell types in a way that seems quite unpredictable. This is obvious from Table 1 which summarizes the cell distribution of some of the surface components of rat lymphocytes, including most of the predominant cell surface molecules. It seems unlikely from the patterns of expression that any of the molecules in Table 1 are involved in metabolic functions of lymphocytes. Molecules involved in metabolite transport would probably be present in low numbers on resting lymphocytes, since these cells are almost inert in biosynthetic functions. It might be argued that the strange patterns summarized in Table 1 are an artifact of using antibodies (and particularly monoclonal antibodies) to study the tissue distributions of membrane molecules. Any one antibody reacts with only a small part of an antigen and unexpected cross-reactions could occur if antibodies are multispecific (Richards et al., 1975) or if a small portion of surface structure was similar between otherwise unrelated antigens. With glycoproteins there is the possibility of the same carbohydrate structure being found on different proteins. However, artifacts are not likely to be the general rule since a number of the antigens with unusual distributions have been isolated from quite different cell types and shown to be structurally very similar and possibly identical at least in the polypeptide part of the molecule. These include Thy-l antigen of brain, thymus and fibroblasts (Barclay et al., 1976; Cotmore, Crowhurst & Waterfield, 1981); class II histocompatibility antigen of B lymphocytes and epithelial cells (Klareskog, Forsum & Peterson, 1980); W3/13 antigen of thymocytes and neutrophils (Standring et al., 1978) and MRC OX 2 antigen of thymocytes and brain (A. N. Barclay & H. Ward, unpublished observations). The only well-established case of an apparently irrelevant cross-reaction involving natural antigens is that of the monoclonal antibody which reacts with Thy-l antigen and vimentin (Dulbecco et af., 1981). Functions are known for only three of the molecules in Table 1, namely cell surface Ig and class I and II histocompatibility antigens. Ig is the antigen receptor of B lymphocytes and accordingly is synthesised only by this cell
SURFACE
MOLECULES
AND
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INTERACTIONS
225
type (Warner, 1974). It fits with the concept of a molecule related to the differentiated state of the cell. The functions of class I and II histocompatibility antigens are less clearly defined but it is known that they are controlling or restricting elements in T lymphocyte recognition. T cells appear to recognise antigen only if it is presented on a cell surface along with histocompatibility antigen. The part of the histocompatibility antigen which is recognised is that which shows polymorphism within the species (Klein 1979). Thus the histocompatibility antigens are clearly involved in cell-cell interactions between the T cell and the antigen-presenting cell. Class I histocompatibility antigens are recognised by cytotoxic T cells and may be particularly relevant to the killing of cells infected with virus, while class II antigens are recognised by helper T cells and are thus important in the initiation of immune responses controlled by these cells. The wide tissue distribution of class I antigens fits well with its function since most cell types can be infected by viruses. However, the distribution of class II antigens is a puzzle since only macrophages and/or dendritic cells are known to be involved in the presentation of antigen to helper T cells. The Thy-l, W3/13 and MRC OX 2 antigens are extreme examples of antigens appearing on unrelated cell types and these share the property of being expressed on lymphoid cells and in the brain. This relationship seems to be a common one and other brain/lymphoid antigens which are probably different from those in Table 1 have been described in other species (Dalchau, Kirkley & Fabre, 1980; Siodak & Nowinski, 1981; Hogg ef al., 1981). Other tissues do not seem to share membrane antigens with lymptoid cells in a similar way to that seen with brain cells. For example, I know of no antigens which are shared between lymphoid and liver cells yet not widely distributed on other cell types. The reason for the common expression of surface antigens between brain and lymphoid tissues is unknown, but it could be that these molecules mediate cell-cell interactions and that brain and lymphoid cells are more extensively involved in such interactions than most other cell types in mature organisms. The Thy-l antigen shows further unusual characteristics when its expression is examined in different species. Table 2 shows the tissue distribution in those species where a Thy-l homologue has been firmly identified on the basis of serological cross-reactions and structural homology. Expression is conserved in the brain and probably on fibroblasts but not in the immune system. The differences in expression between the closely related species mus and ruttus are striking and in humans there is no Thy-l antigen expressed on thymocytes or T lymphocytes. Other molecules found at lymphoid surfaces also show varying degrees of conservation within or between species. In the mouse two main types
226
A. F. WILLIAMS
2 in the expression of Thy- 1 antigen amongst some mammalian species TABLE
Variation
Species Tissue or cell type Brain (neuronal) Fibroblasts Thymocytes T lymphocytes Bone marrow lymphoid cells Haemopoietic stem cells
Mouse
Rat
Dog
Human
++++ ++ ++++ +
++++ ++ +++i++ ++
++++ n.k. + + + n.k.
+++t
-
++ n.k.
The identification of a homologue of rodent Thy-l is based on antigenic cross-reaction and purification of a molecule with similar structural characteristics (from data reviewed in Campbell et al., 1981 and Cotmore et al., 1981). The number of crosses gives a rough idea of the amount of antigen on different cell types and n.k. means not known.
of class II antigens called the I-A and the I-E/C products are found, yet some mouse strains fail to express the I-E/C product (Jones, Murphy & McDevitt, 1981). Despite this, the predominant class II product in humans is homologous to the I-E/C product of mouse and a human I-A homologue remains to be convincingly demonstrated (Allison et al., 1978). Also in humans but not rats class II antigens are found on the kidney endothelium (Hart et al., 1981). Immunoglobulins also vary between species in the number of sub-classes and also in the functions of some of the classes (Spiegelberg, 1974). Thy-l
Antigen is Homologous
to Immunoglobin
V-Domains
Molecular aspects of brain Thy-l glycoproteins are summarised in Fig. 1 which shows a model for the molecule at the cell surface with the polypeptide folded as for an Ig V-domain (Williams & Gagnon, 1982). The rat Thy-l polypeptide consists of 111 amino acids and has a molecular weight of 12762. There are two disulphide bonds Cys 9-Cys 111 and Cys 19-Cys 85. Asparagine-linked carbohydrate structures are attached at residues 23, 74 and 98 and altogether their molecular weight is about half that of the polypeptide. The Thy- 1 glycoprotein has hydrophobic properties but these cannot be accounted for by the polypeptide. There is strong indirect evidence for a non-protein (presumably lipid) moiety covalently attached to the C-terminal cysteine residue which is likely to attach the molecule to the lipid bilayer (Campbell et al., 1981). The Thy-l glycoprotein from neuronal cells, thymocytes and fibroblasts probably has an identical
SURFACE
MOLECULES
AND
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INTERACTIONS
227
-.~2 “m --& &
_
*-
II c
Gal
Gal
;pid
Non:
protein
toil
2 nm
I
FIG. 1. A model for the Thy-l at the cell surface. The Thy-l polypeptide is shown folded as for the VA Ig domain determined by Edmundson, et al., (1975). The letters A to G indicate the P-strands of the Ig fold also shown schematically in Fig. 3. The bar between strands B and F is the conserved disulphide bond with which the Cys 19-Cys 85 bond of Thy-l is compatible. The bar between strand A and the end of the molecule is drawn to indicate the Cys 9Cys 111 disulphide bond of Thy-l. Other features are: [x1:the positions of N-linked carbohydrate structures at residues 23,74 and 98. Also shown is a typical N-linked carbohydrate structure drawn to scale at top right. Three such structures would cover much of the surface of the molecule. 0: shows the position of the allotype-related sequence difference in mouse Thy-l at residue 89 (Arg/Gln interchange). @: shows places where rat and mouse Thy-l differ by blocks of four and five residues. Much of the rest of the sequence is identical between the species. [From Williams & Gagnon, 19821.
polypeptide but different carbohydrate in each case (Barclay et af., 1976; Cotmore et al., 1981). Also within the Thy-l from thymus there is heterogeneity in the carbohydrate. The Thy-l glycoprotein is the size of an immunoglobulin domain and the disposition of the disulphide bond Cys 19-Cys 85 suggested there may be homology with Ig since this arrangement is similar to the disulphide bond seen in all Ig domains. The Ig domain is a repeating homology unit of about 110 amino acids which has apparently duplicated and diverged in evolution in various ways to make up all the polypeptide chains seen in immunoglobulins (Fig. 2). The domain sequences within Ig heavy and light chains all show a characteristic folding pattern with anti-parallel p-strand secondary structure and almost no cy-helix (Amzel & Poljak, 1979). There are differences between the fold in V- and C-domains with V-domains
228
A.
F. WILLIAMS
n
bM
FIG. 2. Molecules in the Ig superfamily. IgM, class I histocompatibility antigens (HLA-A, B, C) and Thy-l antigen are shown schematically at a cell surface. Ig domains and their homologous regions in Thy-l and HLA-A,B,C, are represented by circles. Intra-chain disulphide bonds are shown by the z symbols and interchain bonds in IgM by dashed lines. N-linked carbohydrate structures are shown by 7 and n and c identify the N-terminus and C-terminus of the polypeptides with the exception of&-m. HLA-A,B,C and IgM are believed to be integrated into the membrane by a hydrophobic protein sequence while for Thy-l the evidence suggests the existence of a non-protein structure for membrane integration. [From Williams & Gagnon, 1982.1
having extra p-strands coming from the centre of the domain as shown in Fig. 3. In looking for homology with immunoglobulins the key characteristics are the conserved disulphide bond, P-sheet structure and sequence homologies. The disulphide bonds of Thy-l both fit perfectly with the Ig fold (Fig. 1) and the molecule has a high content of p-sheet structure as shown by circular dichroism. The sequence homologies strongly suggest that Thy-l will be folded like an Ig variable domain (Williams & Gagnon, 1982). It has prevously been shown that class I histocompatibility antigens have two domains which are homologous to Ig constant region domains. One of these makes up the whole of the µglobulin (& - m) polypeptide and the other consists of 100 residues within the larger polypeptide of class I antigens (Orr et al., 1979). The homology relationships between Thy-l, class I antigens and Ig are summarized in Fig. 2 and it is immediately obvious that Thy-l is a candidate for being like the molecule from which the whole Ig superfamily evolved, since it is the only structure so far described which exists as a single domain
SURFACE
MOLECULES
”
--s--sB C
A \
C
AND
\ ‘A
‘\\ ,+ B
C’ C
CELL
C”D s-y+ D
INTERACTIONS
E
F
F’
G ,,f’
,I’ E
229
G
FIG. 3 Schematic representation of p-strands in Ig V- and C-domains. The top section shows schematically the disposition of segments of P-strands along Ig V- and C-domain sequences. The domains show homologies at the ends of the sequences in strands A,B,C,E,F,G as indicated by the dotted lines. The lower part shows the folding pattern with the two p-sheets laid out and separated by the line down the middle (compare with Fig. 1). For a C-domain, p-strand C continues directly on to P-strand D (dotted line), while a V-domain contains an extra loop of sequence shown by c’ and C”. [Modified from Amzel & Poljak, 1979.1
unassociated with other molecules. Its sequence also suggests similarity to the primordial domain, since it has characteristics which are homologous with conserved elements of both V- and C-domains even though the best fit is with V-domains. A putative homologue of Thy-l has been identified in an invertebrate (the squid) on the basis of partial sequence homology (Williams & Gagnon, 1982). If this is confirmed by the full sequence this finding will further strengthen the possibility that Thy-l is akin to the primordial Ig domain. The molecules shown in Fig. 2 are the only lymphoid membrane molecules for which there is sufficient protein sequence known to allow analysis of homologies with other proteins. The fact that they have all turned out to be related is surprising, since structural studies on each molecule were initiated for reasons other than the possibility of finding homology with Ig. It therefore seems highly likely that other Ig-related membrane molecules will be identified as more sequences are done. It already seems likely that the large polypeptides of TL and Qa-2 antigens will be Ig-related since they are found associated with &-m in a structure similar to the class I antigens (Michaelson et al., 1977).
230
A.
F. WILLIAMS
A key question now is whether or not the structural homologies imply functional homologies. What function could Thy-l glycoprotein have? This question might be rephrased as: What can a single Ig domain do? or: What did the primordial Ig domain do? Functions
of Immunoglobulin
Domains
The functions of immunoglobulins can be divided into antigen recognition by V-domains and mediation of effector functions by C-domains. The antigen combining site is made up of amino acid sequences in the bends connecting p-strands B to C, C’ to C” and F to G as shown in Fig. 3. These loops from both VH and VL domains together contribute to the combining site (Amzel & Poljak, 1979). Thus antigen recognition is a complex phenomenon and also is of little immune consequence without effector mechanisms which result in stimulation of the complement system or the reticula-endothelial system. The effector mechanisms are triggered when various receptor systems bind to C-domains displayed in a multimeric array as a result of the formation of antigen-antibody complexes (Spiegelberg, 1974). Different antibody classes mediate different effector functions and within an antibody molecule different C-domains can mediate different functions. For example, the Clq component of complement recognises the Cn2 domain of IgG (Colomb & Porter, 1975) while the IgG receptor of K cells recognises the Cn3 domain (MacLennan, Connell & Gotch, 1974). Altogether the various C-domains of all the Ig classes and the effector molecules which recognise them can be thought of as a moderately large set of ligand (Ig-domain) : receptor systems involved in triggering events after antigen binding. The interest of this set is that the triggering is basically simple and, at least in some cases, seems restricted to the recognition of a single domain. In seeking the essence of the functions of Ig domains it can be argued that for both V- and C-domains the Ig-fold can be thought of as providing a stable platform on which determinants can be displayed (see Fig. 3). In antibodies these are the amino acids at the bends or on the outer surfaces of the p-sheets. Different sequences in different domains give rise to the various specificities in V- or C-domain functions. On structural grounds McLachlan (1980) has argued that the primordial Ig-domain should have a folding pattern like a V-domain but this need not imply a function similar to antigen recognition. A structure with a V-domain fold could easily be used in simple triggering functions in a manner similar to that seen with Ig C-domains.
SURFACE
MOLECULES
AND
CELL
INTERACTIONS
Hypothesis for the Function of Thy-l end for a Set of &-Related in Cell Interactions
231 Molecules
In thinking of possible functions for Thy-l three points must be taken into account. (1) The structural data and the homology with Ig. (2) The tissue distributions. (3) The variation of expression in evolution. Given the structural results one obvious possibility would be for Thy-l to act as a ligand triggering interactions between cells. For example, Thy-l may be the sort of molecule which could mediate recognition between neuronal and glial cells in the brain. The molecule could be thought of as functioning in a manner analogous to Ig C-domains in their interactions with the receptors of the Ig effector systems. More generally the following points are postulated (Cohen et al., 1981). (1) There is a family of molecules related to Ig which function to trigger cell-cell interactions. More Ig-related molecules than those shown in Fig. 2 should be found when more cell surface molecules are sequenced. (2) There are receptors (so far unidentified) for the Ig-related molecules and these will have co-evolved along with the molecules they recognise. This set of ligand-receptor systems is analogous to the C-domains of Ig and their receptors. (3) The Ig-related molecules like Thy-l pre-date antibodies and histocompatibility antigens, since this set of molecules evolved to trigger initiation and cessation of cell movement and/or differentiation. Thy-l cannot be thought of as having a role particularly to do with immunity since its expression is not conserved in lymphoid cells (Table 2). If a set of Ig-related molecules evolved along with the evolution of multi-cellular organisms then this would provide ample raw material for the evolution of immunoglobulins and the major histocompatibility antigens whirl so far have been found only in vertebrates (NisonofI, Hopper & Spring, 1975; Cohen, 1980). (4) Any particular molecule of the set involved in cell-cell interactions can function in more than one type of tissue. This is suggested to account for the unpredictable tissue distributions of cell surface molecules (Table 1). To have specific interactions there is no need for a unique ligand : receptor system for each set of interacting cell types. In many cases different cell types are separated either anatomically or temporally in ontogeny and the same triggering system could be used for different interactions without inappropriate reactions occurring. If the same triggering system is used for different cells then the functions of the triggered cells must be a consequence of their differentiated state, not of the triggering systems. The set of triggering molecules can be thought of as functionally equivalent and the
232
A.
F. WILLIAMS
only reason for one to be used rather than another would be because of the way things evolved. If this is so a change from one 1igand:receptor pair to another may account for the lack of conservation of antigen expression in different species (Table 2). The difficulty in this is that a co-ordinated change of expression of ligand and receptor on different cell types would have to occur. Determinants
on Thy-l
Which may be Recognised
If one postulates that a receptor for Thy-l exists it is interesting to consider which parts of the Thy-l glycoprotein might be recognised. From the model in Fig. 1 it can be suggested that the loops of protein sequence pointing away from the membrane are accessible to interaction with other proteins. This is supported by the fact that the allo-antigenic determinant of mouse Thy-l seems to be determined by an Arg-Gln interchange on one of these loops (Williams & Gagnon, 1982). This end of the molecule would thus seem a prime site for interaction with a receptor. However, two of these loops of sequence show extreme variability between rat and mouse which would argue against a conserved recognition function. Alternatively one could argue that these patches of variability are significant and that there is a co-evolving receptor-determinant system which actually selects for variation in the determinants recognised. This may seem perverse but it is the polymorphic part of histocompatibility antigens which is responsible for the phenomenon of restriction in T-cell interactions (Zinkernagel & Doherty, 1974) and this might argue for receptor systems coevolving with determinants which are varying in evolution. Another alternative is that the determinants recognised on Thy-l are the carbohydrates and that thus the specificities are actually different on different tissues. A role for the carbohydrate in cell interactions was the first function suggested for Thy-l (Barclay et al., 1976) and while the sequence homology with Ig puts emphasis on the protein part of the molecule it does not rule out the possibility that the business end is carbohydrate. Other Molecular
Families
It is not likely that all cell surface molecules with unpredictable tissue distributions will be Ig-related. It is already known that the W3/13 antigen (Table 1) is not in this category. This molecule is a sialoglycoprotein which appears similar to glycophorin of human erythrocytes (Brown et al., 1981). Human glycophorins include at least two related gene products (Furthmayr,
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1978) and it could be that the sialoglycoproteins are another family of molecules which are particularly involved in cell surface functions and have evolved by gene duplication and divergence. It is clear from studies on proteases, globins and other well-characterized protein families that molecules with similar functions are often related in evolution as shown by sequence homology (Dayhoff, Lunt, McLaughlin & Barker, 1972). It will be interesting to see how many new groups of molecules are identified as the cell surface proteins are sequenced. I am grateful to Christine Scott and Gaynor Newton for assistance in the preparation of the manuscript. REFERENCES ALLISON, J. P., WALKER, L. E., RUSSELL, W. A., PELLEGRINO, M. A., FERRONE, S., REISFELD, R. A., FRELINGER, J. A. & SILVER, J. (1978). Proc. natn. Acad. Sci. U.S.A.
75,3953. AMZEL, L. M. & POL.JAK, R. J. (1979). Ann. Rev. Biochem. 48,961. BARCLAY, A. N. (1981a) Immunology 42,591. BARCLAY, A. N. (1981b). Immunology 44,727. BARCLAY, A. N., LETARTE-MUIRHEAD, M., WILLIAMS, A. F. & FAULKES. R. (1976). Nature 263,563. BJ~RCK. L., AKERSTR~M, B. & BERGGARD, I. (1979). Eur. J. Zmmunol. 9,486. BODMER, W. F. (1972). Nature 237.139. BRONNER-FRASER, M. E. & COHEN, A. M. (1980). In Current Topics in Developmental Biology. Vol. 15 (Kevin Hunt, R. ed.). New York: Academic Press. pp. l-25. BROWN, W. R. A., BARCI.AY. A. N., SUNDERLAND, C. A. & WILLIAMS, A. F. (1981). Nature 289,456. BURNET, F. M. (1970). Nature 226, 123. CAMPBELL, D. G., GAGNON, J., REID, K. B. M. & WILLIAMS, A. F. (1981). Rio&em. J.
195, 15. COHEN, A. (1980). In Contemporary Topics in Zmmunobiology Vol. 9. (Marchslonis, J. M. & Cohen, N. eds). New York: Plenum Press. pp. 109-139. COHEN, F. E., NOVOTN~, J., STERNBERG. M. J. E., CAMPBELL, D. G. & WILLIAMS, A. F. (1981). Rio&em. J. 195,31. COLOMB, M. & PORTER, R. R. (1975). Biochem. J. 145, 177. COTMORE, S. F., CROWHURST, S. A. & WATERFIELD. M. D. (1981). Eur. J. Zmmunol. 11,
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DALCHAU, R., KIRKLEY, J. & FABRE, J. W. (1980). Eur. J. Zmmunol. 10,745. DAYHOFF, M. O., HUNT. L. T., MCLAUGHLIN, P. J. & BARKER, W. C. (1972). In Atlas of Protein Sequence and Structure. Vol. 5 (Dayhoff, M. 0. ed.). The National Biomedical Research Foundation, P. 0. Box 629, Silver Spring, Maryland 20901, U.S.A. DULBECCO, R., UNGER, M., BOLOGNA, M., BATTIFORA, H., SYKA. P. & OKADA, S. (1981). Nature 292, 772. EDMUNDSON, A. B., ELY, K. R., ABOLA, E. E., SCHIFFER, M. & PANAGIOTOPOULOS, N. (1975). Biochemistry 14,3953. FORD, W. L., SMITH. M. E. & ANDREWS, P. (1978). In Cell-Cell Recognition (Curtis A. S. G. , ed). Cambridge: Cambridge University Press. pp. 359-392. FURTHMAYR, H. (1978). Nature 271, 519. GALLY, J. A. & EDELMAN, G. M. (1972). Ann. Rev. Genet. 6, 1.
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HART, D. N. .I., FUGGLE, S. V., WILLIAMS, K. A., FABRE, J. W., TING. A. & MORRIS, P. J. (1981). Transplant&on 31,428. HOGG, N., SLUSARENKO, M., COHEN, J. & REISNER, J. (1981). Ceil 24,875. HOOD, L. E., HUANG, H. V. & DREYER. W. J. (1977). J. Supramolecular Structure 7, 531. HOOD, L. E., WEISMANN, I. L. & WOOD, W. B. (1978). Immunology. Menlo Park: The Benjamin/Cummings Publishing Co. JONES, P. P., MURPHY, D. B. & MCDEVITT, H. 0. (1981). Immunogenetics 12,321. KLARESKOG, L., FORSUM. U. & PETERSON, P. A. (1980). Eur. J. Zmmunol. 10,958. KLEIN, J. (1979). Science 203, 516. MCLACHLAN, A. D. (1980). In Proteins and Related Subjects. 28, Protides of The Biological Fluids (Peeters, H. , ed.). Oxford: Pergamon Press. pp. 29-32. MACLENNAN, I. C. M., CONNELL, G. E. & GOTCH. F. M. (1974). Immunology 26,303. MICHAELSON, J., FLAHERTY, L., VITTETTA, E & POULIK, M. D. (1977). J. exp. Med. 145, 1066. NISSONOFF, A., HOPPER, J. E. & SPRING, S. B. (1975). The Antibody Molecule. New York: Academic Press. ORR, H. T., LANCET, D., ROBB, R. J., LOPEZ DE CASTRO, J. A. & STROMINGER, J. L. (1979). Nature 282,266. RICHARDS, F. F., KONIGSBERG, W. H., ROSENSTEIN, R. W. & VARGA, J. M. (1975). Science 187, 130. SIODAK, A. W. & NOWINSKI, R. C. (1981). Immunogenetics 12,45. SPIEGELBERG, H. L. (1974). Ado. Zmmunol. 19,259. STANDRING, R., MCMASTER, W. R., SUNDERLAND, C. A. SC WILLIAMS, A. F. (1978). Eur. J. Zmmunol. 8,832. THIELE, H-G., ARNDT, R., STARK, R. & WONIGEIT, K. (1979). Transplantation Proc. XI, 1636. WARNER. N. L. (1974). Adv. Zmmunol. 19,67. WILLIAMS, A. F. (1980). Biochem. Sot. Symp. 45,27. WILLIAMS, A. F. & GAGNON, J. (1982). Science, 216.696. WILLIAMS, A. F., GALFRI?:. G. &MIL&EIN, C. i197?‘). Cell 12,663. ZINKERNAGEL, R. M. & DOHERTY: P. C. (1974) Nature (London) 251,547.
Surface Molecules and Cell Interactions ALAN F. WILLIAMS MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX 1 3RE England (Received
10 February
1982)
Many of the cell surface molecules of lymphocytes or their precursors are expressed in an unpredictable way on a limited set of other cell types. This often seems to involve expression on lymphoid and brain cells. The Thy-l antigen is in this category, being a major glycoprotein of murine neuronal cells, fibroblasts and thymocytes. Structural studies show that this molecule is homologous with immunoglobulin domains which are the structural sub-units of all immunoglobulin polypeptides. Thy-l is the size of one immunoglobulin domain and its sequence is most homologous with variable regions of immunoglobulins. It is suggested that Thy-l is one of a set of surface molecules concerned with triggering interactions between cells and that this is the primitive function of the immunoglobulin domain. Cell interactions could be medi-
ated by domain-like
structures and receptors for them in a way which
parallels the triggering of immunological effector reactions by the interaction of receptors with immunoglobulin constant regions. If this is so then the structure seen in the immunoglobulin domain would have evolved along with the evolution of cell organisation. The genes specifying the cell interaction molecules could then have provided the genetic material for the evolution of antibody and histocompatibility antigen at the time of vertebrate emergence.
Lymphocyte
Functions
and the Cell Surface
It seems highly likely that molecules on adjacent cell surfaces interact to influence the behaviour of cells. Amongst other things, such interactions
could provide start or stop signals for cell movement and essential triggers for division and differentiation of cells at various stages in their lineages. Cues for cell movement and differentiation are needed during embryogenesis (Bronner-Fraser & Cohen, 1980) and also in many aspects of the behaviour of cells in adult organisms. The lymphocyte is a good example of a cell type which is continuously involved in various interactions with other cells. In its resting state the 221
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Press Inc. (London)
Ltd.
222
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WILLIAMS
lymphocyte recirculates from blood to lymph until being triggered to divide and differentiate by antigen. Lymphocyte recirculation involves specific recognition of specialised endothelium in lymph nodes and migration between the endothelial cells into positions which are different for B and T lymphocytes (Ford, Smith & Andrews, 1978). To mediate this behaviour there must be triggers for adhesion, movement and localisation of lymphocytes. The interaction of lymphocytes with antigen also involves cell-cell interactions since T lymphocytes appear to recognise antigen at the cell surface of macrophages (or another similar cell type) and B lymphocytes probably interact with macrophages and T lymphocytes in the process of being stimulated by antigen (basic text Hood, Weissman & Wood, 1978). These interactions with antigen and other cell types result in cell division and entry of lymphocytes into a new phase of differentiation. All these functions are of interest in their own right, but they may also provide a model for movement and differentiation of other cell types. As well as being functionally interesting there are technical reasons why lymphocytes are more suitable for cell surface studies than most other cell types. Lymphocytes undertake all their interactions without forming irreversible junctions with other cells and thus can be isolated without the use of proteolytic or other degradative enzymes. Large numbers of cells can be obtained (>lO”) and this allows the purification of membrane molecules for structural and functional studies. The molecules found at the surface of thymocytes and T and B lymphocytes are now being characterized. This is mostly being done by preparing monoclonal antibodies against mixtures of membrane molecules and then using the antibodies to identify the constituents of the membrane (Williams, Galfre & Milstein, 1977; Williams, 1980). As the molecules are first indentified with antibodies they are initially referred to as antigens and often by the arbitrary name of the monoclonal antibody used for their identification (Table 1). The studies on lymphoid membrane molecules are at an early stage but there have already been surprises. The unpredictable distribution of lymphocyte antigens on other cells presents one puzzle and unexpected structural homologies between surface antigens and immunoglobulins (Ig) present another. These may be reconciled in an hypothesis which suggests that there is a set of structures, including Ig-related molecules, which mediate cell interactions and can function in a similar way on various cell types. Others have speculated on related themes (Burnet, 1970; Bodmer, 1972; Gally & Edelman, 1972; Hood, Huang & Dreyer, 1977) but previous ideas will not be reviewed in this paper.
:
antigens
antigens:
antigen
antigen
:
lymphocytes
2 X 55 000 gp 2x25 ooogp
30 000 gp 28 000 gp
43 000 gp 12 000 p
nature mol. wt. (gp) protein (p)
21000p
gP
2 x lo4 thymocytes not known
gP
not known
47 000 gp
17500-187OOgp
95 000 gp
170000-220000gp
tetramer:
dimer:
dimer:
Molecular glycoprotein
3 x 10” thymocytes
1 S x lo4 thymocytes
2 x lo4 thymocytes
lo6 thymocytes
10’ thymocytes
7 x lo4 thymocytes
5 x 104B lymphocytes
1.5 x lo58
10’ lymphocytes
Sites per cell on cell named on’
only.
or
T lymphocytes.
Thymocytes, brain, follicular dendritic cells, endothelial cells. Thymocytes, T lymphocyte sub-set, macrophages Thymocytes, T lymphocyte sub-set, natural killer cCIIS. Thymocytes, all T lymphocytes.
Most haemopoietic cells with different carbohydrate on different cell types. Thymocytes, T lymphocytes, polymorphs, plasma cells, brain Thymocytes, neuronal cells, fibroblasts, connective tissue, myoblasts, immature B lymphocytes and haemopoietic stem cells.
B lymphocytes
Many cell types but not neuronal glial cells, also very low on most thymocytes. B lymphocytes, dendritic cells, activated macrophages, epithelial cells of various organs.
Expressed
t Data from references reviewed in Williams (1980); Brown et al. (1981); Campbell er al. (1981) plus additional data from Bjiirck, AkerstrGm % Berggird (1979); Thiele, Arndt, Stark & Wonigeit (1979); Barclay (1981a) and (19816) and Barclay, A. N., Dallman, M. & Ward, H., unpublished observations. D Antigen is present on the named cell types and not found on other cell types seen in tissue sections of lymphoid organs, kidney, brain. gut or liver.
Pta (Ly-2)
OX
MRC
19 antigen
OX 8 antigen
antigen
MRC
W3/25
Minor glycoproteins and antigens MRC OX 2 antigen
Leucocyte sialoglycoprotein (W3/13 antigen) Thy-l antigen
Leucocyte-common
Major glycoproreins
Immunoglobulin:
Class II
Major histocompafibility Class I
Cell surface
1
Some cell surface antigens of rat lymphocytes?
TABLE
z W
224
A.
The Tissue Distribution
F.
WILLIAMS
of Lymphocyte
Cell Surface Molecules
The pattern of distribution on other cell types of molecules (antigens) found at the lymphocyte surface is surprising. Arguing on a simple-minded basis it might be expected that cell-surface molecules would either be widely distributed on most cells or tightly restricted to one or a few functionally related cell types. Molecules involved in basic metabolic functions would be widely distributed and those concerned with the specialised functions of cells restricted. For lymphocytes most surface molecules do not fit into either of these categories but rather are found on various but not all cell types in a way that seems quite unpredictable. This is obvious from Table 1 which summarizes the cell distribution of some of the surface components of rat lymphocytes, including most of the predominant cell surface molecules. It seems unlikely from the patterns of expression that any of the molecules in Table 1 are involved in metabolic functions of lymphocytes. Molecules involved in metabolite transport would probably be present in low numbers on resting lymphocytes, since these cells are almost inert in biosynthetic functions. It might be argued that the strange patterns summarized in Table 1 are an artifact of using antibodies (and particularly monoclonal antibodies) to study the tissue distributions of membrane molecules. Any one antibody reacts with only a small part of an antigen and unexpected cross-reactions could occur if antibodies are multispecific (Richards et al., 1975) or if a small portion of surface structure was similar between otherwise unrelated antigens. With glycoproteins there is the possibility of the same carbohydrate structure being found on different proteins. However, artifacts are not likely to be the general rule since a number of the antigens with unusual distributions have been isolated from quite different cell types and shown to be structurally very similar and possibly identical at least in the polypeptide part of the molecule. These include Thy-l antigen of brain, thymus and fibroblasts (Barclay et al., 1976; Cotmore, Crowhurst & Waterfield, 1981); class II histocompatibility antigen of B lymphocytes and epithelial cells (Klareskog, Forsum & Peterson, 1980); W3/13 antigen of thymocytes and neutrophils (Standring et al., 1978) and MRC OX 2 antigen of thymocytes and brain (A. N. Barclay & H. Ward, unpublished observations). The only well-established case of an apparently irrelevant cross-reaction involving natural antigens is that of the monoclonal antibody which reacts with Thy-l antigen and vimentin (Dulbecco et af., 1981). Functions are known for only three of the molecules in Table 1, namely cell surface Ig and class I and II histocompatibility antigens. Ig is the antigen receptor of B lymphocytes and accordingly is synthesised only by this cell
SURFACE
MOLECULES
AND
CELL
INTERACTIONS
225
type (Warner, 1974). It fits with the concept of a molecule related to the differentiated state of the cell. The functions of class I and II histocompatibility antigens are less clearly defined but it is known that they are controlling or restricting elements in T lymphocyte recognition. T cells appear to recognise antigen only if it is presented on a cell surface along with histocompatibility antigen. The part of the histocompatibility antigen which is recognised is that which shows polymorphism within the species (Klein 1979). Thus the histocompatibility antigens are clearly involved in cell-cell interactions between the T cell and the antigen-presenting cell. Class I histocompatibility antigens are recognised by cytotoxic T cells and may be particularly relevant to the killing of cells infected with virus, while class II antigens are recognised by helper T cells and are thus important in the initiation of immune responses controlled by these cells. The wide tissue distribution of class I antigens fits well with its function since most cell types can be infected by viruses. However, the distribution of class II antigens is a puzzle since only macrophages and/or dendritic cells are known to be involved in the presentation of antigen to helper T cells. The Thy-l, W3/13 and MRC OX 2 antigens are extreme examples of antigens appearing on unrelated cell types and these share the property of being expressed on lymphoid cells and in the brain. This relationship seems to be a common one and other brain/lymphoid antigens which are probably different from those in Table 1 have been described in other species (Dalchau, Kirkley & Fabre, 1980; Siodak & Nowinski, 1981; Hogg ef al., 1981). Other tissues do not seem to share membrane antigens with lymptoid cells in a similar way to that seen with brain cells. For example, I know of no antigens which are shared between lymphoid and liver cells yet not widely distributed on other cell types. The reason for the common expression of surface antigens between brain and lymphoid tissues is unknown, but it could be that these molecules mediate cell-cell interactions and that brain and lymphoid cells are more extensively involved in such interactions than most other cell types in mature organisms. The Thy-l antigen shows further unusual characteristics when its expression is examined in different species. Table 2 shows the tissue distribution in those species where a Thy-l homologue has been firmly identified on the basis of serological cross-reactions and structural homology. Expression is conserved in the brain and probably on fibroblasts but not in the immune system. The differences in expression between the closely related species mus and ruttus are striking and in humans there is no Thy-l antigen expressed on thymocytes or T lymphocytes. Other molecules found at lymphoid surfaces also show varying degrees of conservation within or between species. In the mouse two main types
226
A. F. WILLIAMS
2 in the expression of Thy- 1 antigen amongst some mammalian species TABLE
Variation
Species Tissue or cell type Brain (neuronal) Fibroblasts Thymocytes T lymphocytes Bone marrow lymphoid cells Haemopoietic stem cells
Mouse
Rat
Dog
Human
++++ ++ ++++ +
++++ ++ +++i++ ++
++++ n.k. + + + n.k.
+++t
-
++ n.k.
The identification of a homologue of rodent Thy-l is based on antigenic cross-reaction and purification of a molecule with similar structural characteristics (from data reviewed in Campbell et al., 1981 and Cotmore et al., 1981). The number of crosses gives a rough idea of the amount of antigen on different cell types and n.k. means not known.
of class II antigens called the I-A and the I-E/C products are found, yet some mouse strains fail to express the I-E/C product (Jones, Murphy & McDevitt, 1981). Despite this, the predominant class II product in humans is homologous to the I-E/C product of mouse and a human I-A homologue remains to be convincingly demonstrated (Allison et al., 1978). Also in humans but not rats class II antigens are found on the kidney endothelium (Hart et al., 1981). Immunoglobulins also vary between species in the number of sub-classes and also in the functions of some of the classes (Spiegelberg, 1974). Thy-l
Antigen is Homologous
to Immunoglobin
V-Domains
Molecular aspects of brain Thy-l glycoproteins are summarised in Fig. 1 which shows a model for the molecule at the cell surface with the polypeptide folded as for an Ig V-domain (Williams & Gagnon, 1982). The rat Thy-l polypeptide consists of 111 amino acids and has a molecular weight of 12762. There are two disulphide bonds Cys 9-Cys 111 and Cys 19-Cys 85. Asparagine-linked carbohydrate structures are attached at residues 23, 74 and 98 and altogether their molecular weight is about half that of the polypeptide. The Thy- 1 glycoprotein has hydrophobic properties but these cannot be accounted for by the polypeptide. There is strong indirect evidence for a non-protein (presumably lipid) moiety covalently attached to the C-terminal cysteine residue which is likely to attach the molecule to the lipid bilayer (Campbell et al., 1981). The Thy-l glycoprotein from neuronal cells, thymocytes and fibroblasts probably has an identical
SURFACE
MOLECULES
AND
CELL
INTERACTIONS
227
-.~2 “m --& &
_
*-
II c
Gal
Gal
;pid
Non:
protein
toil
2 nm
I
FIG. 1. A model for the Thy-l at the cell surface. The Thy-l polypeptide is shown folded as for the VA Ig domain determined by Edmundson, et al., (1975). The letters A to G indicate the P-strands of the Ig fold also shown schematically in Fig. 3. The bar between strands B and F is the conserved disulphide bond with which the Cys 19-Cys 85 bond of Thy-l is compatible. The bar between strand A and the end of the molecule is drawn to indicate the Cys 9Cys 111 disulphide bond of Thy-l. Other features are: [x1:the positions of N-linked carbohydrate structures at residues 23,74 and 98. Also shown is a typical N-linked carbohydrate structure drawn to scale at top right. Three such structures would cover much of the surface of the molecule. 0: shows the position of the allotype-related sequence difference in mouse Thy-l at residue 89 (Arg/Gln interchange). @: shows places where rat and mouse Thy-l differ by blocks of four and five residues. Much of the rest of the sequence is identical between the species. [From Williams & Gagnon, 19821.
polypeptide but different carbohydrate in each case (Barclay et af., 1976; Cotmore et al., 1981). Also within the Thy-l from thymus there is heterogeneity in the carbohydrate. The Thy-l glycoprotein is the size of an immunoglobulin domain and the disposition of the disulphide bond Cys 19-Cys 85 suggested there may be homology with Ig since this arrangement is similar to the disulphide bond seen in all Ig domains. The Ig domain is a repeating homology unit of about 110 amino acids which has apparently duplicated and diverged in evolution in various ways to make up all the polypeptide chains seen in immunoglobulins (Fig. 2). The domain sequences within Ig heavy and light chains all show a characteristic folding pattern with anti-parallel p-strand secondary structure and almost no cy-helix (Amzel & Poljak, 1979). There are differences between the fold in V- and C-domains with V-domains
228
A.
F. WILLIAMS
n
bM
FIG. 2. Molecules in the Ig superfamily. IgM, class I histocompatibility antigens (HLA-A, B, C) and Thy-l antigen are shown schematically at a cell surface. Ig domains and their homologous regions in Thy-l and HLA-A,B,C, are represented by circles. Intra-chain disulphide bonds are shown by the z symbols and interchain bonds in IgM by dashed lines. N-linked carbohydrate structures are shown by 7 and n and c identify the N-terminus and C-terminus of the polypeptides with the exception of&-m. HLA-A,B,C and IgM are believed to be integrated into the membrane by a hydrophobic protein sequence while for Thy-l the evidence suggests the existence of a non-protein structure for membrane integration. [From Williams & Gagnon, 1982.1
having extra p-strands coming from the centre of the domain as shown in Fig. 3. In looking for homology with immunoglobulins the key characteristics are the conserved disulphide bond, P-sheet structure and sequence homologies. The disulphide bonds of Thy-l both fit perfectly with the Ig fold (Fig. 1) and the molecule has a high content of p-sheet structure as shown by circular dichroism. The sequence homologies strongly suggest that Thy-l will be folded like an Ig variable domain (Williams & Gagnon, 1982). It has prevously been shown that class I histocompatibility antigens have two domains which are homologous to Ig constant region domains. One of these makes up the whole of the µglobulin (& - m) polypeptide and the other consists of 100 residues within the larger polypeptide of class I antigens (Orr et al., 1979). The homology relationships between Thy-l, class I antigens and Ig are summarized in Fig. 2 and it is immediately obvious that Thy-l is a candidate for being like the molecule from which the whole Ig superfamily evolved, since it is the only structure so far described which exists as a single domain
SURFACE
MOLECULES
”
--s--sB C
A \
C
AND
\ ‘A
‘\\ ,+ B
C’ C
CELL
C”D s-y+ D
INTERACTIONS
E
F
F’
G ,,f’
,I’ E
229
G
FIG. 3 Schematic representation of p-strands in Ig V- and C-domains. The top section shows schematically the disposition of segments of P-strands along Ig V- and C-domain sequences. The domains show homologies at the ends of the sequences in strands A,B,C,E,F,G as indicated by the dotted lines. The lower part shows the folding pattern with the two p-sheets laid out and separated by the line down the middle (compare with Fig. 1). For a C-domain, p-strand C continues directly on to P-strand D (dotted line), while a V-domain contains an extra loop of sequence shown by c’ and C”. [Modified from Amzel & Poljak, 1979.1
unassociated with other molecules. Its sequence also suggests similarity to the primordial domain, since it has characteristics which are homologous with conserved elements of both V- and C-domains even though the best fit is with V-domains. A putative homologue of Thy-l has been identified in an invertebrate (the squid) on the basis of partial sequence homology (Williams & Gagnon, 1982). If this is confirmed by the full sequence this finding will further strengthen the possibility that Thy-l is akin to the primordial Ig domain. The molecules shown in Fig. 2 are the only lymphoid membrane molecules for which there is sufficient protein sequence known to allow analysis of homologies with other proteins. The fact that they have all turned out to be related is surprising, since structural studies on each molecule were initiated for reasons other than the possibility of finding homology with Ig. It therefore seems highly likely that other Ig-related membrane molecules will be identified as more sequences are done. It already seems likely that the large polypeptides of TL and Qa-2 antigens will be Ig-related since they are found associated with &-m in a structure similar to the class I antigens (Michaelson et al., 1977).
230
A.
F. WILLIAMS
A key question now is whether or not the structural homologies imply functional homologies. What function could Thy-l glycoprotein have? This question might be rephrased as: What can a single Ig domain do? or: What did the primordial Ig domain do? Functions
of Immunoglobulin
Domains
The functions of immunoglobulins can be divided into antigen recognition by V-domains and mediation of effector functions by C-domains. The antigen combining site is made up of amino acid sequences in the bends connecting p-strands B to C, C’ to C” and F to G as shown in Fig. 3. These loops from both VH and VL domains together contribute to the combining site (Amzel & Poljak, 1979). Thus antigen recognition is a complex phenomenon and also is of little immune consequence without effector mechanisms which result in stimulation of the complement system or the reticula-endothelial system. The effector mechanisms are triggered when various receptor systems bind to C-domains displayed in a multimeric array as a result of the formation of antigen-antibody complexes (Spiegelberg, 1974). Different antibody classes mediate different effector functions and within an antibody molecule different C-domains can mediate different functions. For example, the Clq component of complement recognises the Cn2 domain of IgG (Colomb & Porter, 1975) while the IgG receptor of K cells recognises the Cn3 domain (MacLennan, Connell & Gotch, 1974). Altogether the various C-domains of all the Ig classes and the effector molecules which recognise them can be thought of as a moderately large set of ligand (Ig-domain) : receptor systems involved in triggering events after antigen binding. The interest of this set is that the triggering is basically simple and, at least in some cases, seems restricted to the recognition of a single domain. In seeking the essence of the functions of Ig domains it can be argued that for both V- and C-domains the Ig-fold can be thought of as providing a stable platform on which determinants can be displayed (see Fig. 3). In antibodies these are the amino acids at the bends or on the outer surfaces of the p-sheets. Different sequences in different domains give rise to the various specificities in V- or C-domain functions. On structural grounds McLachlan (1980) has argued that the primordial Ig-domain should have a folding pattern like a V-domain but this need not imply a function similar to antigen recognition. A structure with a V-domain fold could easily be used in simple triggering functions in a manner similar to that seen with Ig C-domains.
SURFACE
MOLECULES
AND
CELL
INTERACTIONS
Hypothesis for the Function of Thy-l end for a Set of &-Related in Cell Interactions
231 Molecules
In thinking of possible functions for Thy-l three points must be taken into account. (1) The structural data and the homology with Ig. (2) The tissue distributions. (3) The variation of expression in evolution. Given the structural results one obvious possibility would be for Thy-l to act as a ligand triggering interactions between cells. For example, Thy-l may be the sort of molecule which could mediate recognition between neuronal and glial cells in the brain. The molecule could be thought of as functioning in a manner analogous to Ig C-domains in their interactions with the receptors of the Ig effector systems. More generally the following points are postulated (Cohen et al., 1981). (1) There is a family of molecules related to Ig which function to trigger cell-cell interactions. More Ig-related molecules than those shown in Fig. 2 should be found when more cell surface molecules are sequenced. (2) There are receptors (so far unidentified) for the Ig-related molecules and these will have co-evolved along with the molecules they recognise. This set of ligand-receptor systems is analogous to the C-domains of Ig and their receptors. (3) The Ig-related molecules like Thy-l pre-date antibodies and histocompatibility antigens, since this set of molecules evolved to trigger initiation and cessation of cell movement and/or differentiation. Thy-l cannot be thought of as having a role particularly to do with immunity since its expression is not conserved in lymphoid cells (Table 2). If a set of Ig-related molecules evolved along with the evolution of multi-cellular organisms then this would provide ample raw material for the evolution of immunoglobulins and the major histocompatibility antigens whirl so far have been found only in vertebrates (NisonofI, Hopper & Spring, 1975; Cohen, 1980). (4) Any particular molecule of the set involved in cell-cell interactions can function in more than one type of tissue. This is suggested to account for the unpredictable tissue distributions of cell surface molecules (Table 1). To have specific interactions there is no need for a unique ligand : receptor system for each set of interacting cell types. In many cases different cell types are separated either anatomically or temporally in ontogeny and the same triggering system could be used for different interactions without inappropriate reactions occurring. If the same triggering system is used for different cells then the functions of the triggered cells must be a consequence of their differentiated state, not of the triggering systems. The set of triggering molecules can be thought of as functionally equivalent and the
232
A.
F. WILLIAMS
only reason for one to be used rather than another would be because of the way things evolved. If this is so a change from one 1igand:receptor pair to another may account for the lack of conservation of antigen expression in different species (Table 2). The difficulty in this is that a co-ordinated change of expression of ligand and receptor on different cell types would have to occur. Determinants
on Thy-l
Which may be Recognised
If one postulates that a receptor for Thy-l exists it is interesting to consider which parts of the Thy-l glycoprotein might be recognised. From the model in Fig. 1 it can be suggested that the loops of protein sequence pointing away from the membrane are accessible to interaction with other proteins. This is supported by the fact that the allo-antigenic determinant of mouse Thy-l seems to be determined by an Arg-Gln interchange on one of these loops (Williams & Gagnon, 1982). This end of the molecule would thus seem a prime site for interaction with a receptor. However, two of these loops of sequence show extreme variability between rat and mouse which would argue against a conserved recognition function. Alternatively one could argue that these patches of variability are significant and that there is a co-evolving receptor-determinant system which actually selects for variation in the determinants recognised. This may seem perverse but it is the polymorphic part of histocompatibility antigens which is responsible for the phenomenon of restriction in T-cell interactions (Zinkernagel & Doherty, 1974) and this might argue for receptor systems coevolving with determinants which are varying in evolution. Another alternative is that the determinants recognised on Thy-l are the carbohydrates and that thus the specificities are actually different on different tissues. A role for the carbohydrate in cell interactions was the first function suggested for Thy-l (Barclay et al., 1976) and while the sequence homology with Ig puts emphasis on the protein part of the molecule it does not rule out the possibility that the business end is carbohydrate. Other Molecular
Families
It is not likely that all cell surface molecules with unpredictable tissue distributions will be Ig-related. It is already known that the W3/13 antigen (Table 1) is not in this category. This molecule is a sialoglycoprotein which appears similar to glycophorin of human erythrocytes (Brown et al., 1981). Human glycophorins include at least two related gene products (Furthmayr,
SURFACE
MOLECULES
AND
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INTERACTIONS
233
1978) and it could be that the sialoglycoproteins are another family of molecules which are particularly involved in cell surface functions and have evolved by gene duplication and divergence. It is clear from studies on proteases, globins and other well-characterized protein families that molecules with similar functions are often related in evolution as shown by sequence homology (Dayhoff, Lunt, McLaughlin & Barker, 1972). It will be interesting to see how many new groups of molecules are identified as the cell surface proteins are sequenced. I am grateful to Christine Scott and Gaynor Newton for assistance in the preparation of the manuscript. REFERENCES ALLISON, J. P., WALKER, L. E., RUSSELL, W. A., PELLEGRINO, M. A., FERRONE, S., REISFELD, R. A., FRELINGER, J. A. & SILVER, J. (1978). Proc. natn. Acad. Sci. U.S.A.
75,3953. AMZEL, L. M. & POL.JAK, R. J. (1979). Ann. Rev. Biochem. 48,961. BARCLAY, A. N. (1981a) Immunology 42,591. BARCLAY, A. N. (1981b). Immunology 44,727. BARCLAY, A. N., LETARTE-MUIRHEAD, M., WILLIAMS, A. F. & FAULKES. R. (1976). Nature 263,563. BJ~RCK. L., AKERSTR~M, B. & BERGGARD, I. (1979). Eur. J. Zmmunol. 9,486. BODMER, W. F. (1972). Nature 237.139. BRONNER-FRASER, M. E. & COHEN, A. M. (1980). In Current Topics in Developmental Biology. Vol. 15 (Kevin Hunt, R. ed.). New York: Academic Press. pp. l-25. BROWN, W. R. A., BARCI.AY. A. N., SUNDERLAND, C. A. & WILLIAMS, A. F. (1981). Nature 289,456. BURNET, F. M. (1970). Nature 226, 123. CAMPBELL, D. G., GAGNON, J., REID, K. B. M. & WILLIAMS, A. F. (1981). Rio&em. J.
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