Immunology and immunochemistry of glycoconjugates

1703 tein metabolism. I cell disease is a multiple enzyme deficiency including sialidases brought about by defects in the vectoring signals for normal lysosomal location and results in accumulation of all classes of glycoconjugates, while galactosialidosis results from a genetic error in the essential second protein that is required to maintain the normal biological half-life of both/3-galactosidase and sialidase. As shown at the conference, the accumulating storage products are typical substrates for sialidase. It would be surprising if such protective proteins did not occur elsewhere as additional factors for regulating steady-state levels of enzymes and observations on some variants of (x-fucosidase deficiency suggest that, in these cases also, the error lies not in the ability to synthesise the enzyme protein but in maintaining its half-life against excessive proteolysis. Here too, it is suggested that a protective factor may exist. It is perhaps significant that fucose is an alternative to sialic acid as a terminal sugar and also that a-fucosidase like/3-galactosidase shows a tendency to aggregate, a phenomenon which in the latter case seems to be dependent upon the protective protein. Most sialidases require a free hydroxyl group at position 4 and acylation at this loc~tiorL effectively inhibits the action of these enzymes except those of viral origin. In view of this, we may predict that a pathological condition will be sooner or later identified which is associated with accumulation of excess amounts of acylated sialo-conjugates as a result of a

deficiency in the acylesterases described by Schauer. The levels of such enzymes could have important control functions on the rate of catabolism of sialoglycoproteins, since this deacylation would be an essential primary event for initiating exoglycosidase attack. Several new glycosidases are reported. The leech ceramide-glycanase is potentially a valuable tool for the analysis of intact carbohydrate chains from giycosphingolipids. The reason why in some species accumulating oligosaccharides in enzyme defects carry GlcNAc2 and others a single GIcNAc can be explained by species variations in the occurrence of chitobiase, an enzyme described some years ago by Stifling and recently recharacterised./3-Mannosidase is another of the 'core' giycosidases that have been characterised and for which animal and most recently human enzyme deficiency states have been described. We may predict that the in vivo use of a number of the specific inhibitors that are becoming a~:aqable will allow us to induce animal models whereP~y the nature of the pathological symptoms relating to a metabolic block in one enzyme or another may be determined before the natural occurrence of a mutation causing such an error has been identified. More importantly, such tools offer the possibility of oligosaccharide remodelling that will speed our understanding of these specificities in cell-cell interactions.

immunology and immunochemistry of giycoconjugates Jerzy K O g C I E L A K

lnstitut of Hematology, ul. Chocimska 5, 00-957 Warsaw, Poland I have had the privilege to attend all past glycoconjugate symposia and this gives me some perspective when evaluating present accomplishments. In the early days, scientific interest of people working in the field focused on blood group and bacterial antigens and glycosaminoglycans. These areas of research were not accidental because from the very beginning the field attracted scientists interested in structure-function relationships of carbohydrates. Thus scientific curiosity as to the immunochemistry, biosynthesis and genetics of antigens is to a large extent responsible for the development of glycoconjugate research and institutionalization of our symposia. Antigens figured prominently also in this symposium. Present status and future prospects of research on blood group A B H and Lewis substances has been reviewed. New data were reported for distribution and developmental changes in different cells and tis-

sues of blood group immunodominant structures with ABH, I and Lewis activity or of giycosyltransferases involved in their biosyntheses. Novel structural variants of ABH blood group active glycolipids were described. A model was proposed for the release of soluble giycosyltransferase from cells based upon a study of the primary structures of membrane bound and soluble forms of/~-galactoside a-6-sialyltransferase from rat liver. The soluble enzyme was lacking the cytoplasmic and membrane domains of the membrane bound form yet its activity was fully retained. /3-Galactoside a-2-L-fucosyitransferase from human plasma, which is of bone marrow origin, has been extensively purified and found to utilize both type 1 and 2 acceptors without preference to either. Blood group O people were shown to exhibit a residual activity of blood group A gene-specified giycosyl transferase thus supporting earlier claims that the O gene

1704 may not be amorphous. Minor erythrocyte glycoproteins (glycophorins C and D) which carry Gerbich antigenic determinants were shown to be related in that D is a shortened version of C. Another group of antigens investigated were cancer-associated antigens. Studies on this subject were made with the aid of monoclonal antibodies raised against cancer cells or based on previous knowledge, against glycoconjugates which accumulate in cancer cells. Many researchers had practical, diagnostic applications in mind and, for example, examined serum fer the presence of antigens shed by tumor cells into the circulation. Others used monoclonal antibodies to examine tissue distribution of glycoconjugates or to delineate epitopes shared by different glycoconjugates. Monoclonal antibodies were also used for a preparative purpose as a specific part of an affinity column matrix. Autoantibody-producing iymphocytes were isolated from humans, including those suffering from the juvenile form of diabetes mellitus, immortalized with Epstein-Barr virus and cloned. Antibodies produced by the clones were shown to be directed against common, sialic acid containing carbohydrate structures. A complete chemical structure of the membrane apchor region of a single variant of variant surface glycoprotein (VSG) from Trypanosoma brucei was reported. The glycoprotein at its carboxyl-terminus is finked to ethanolamine which in turn is attached through a phosphodiester bond to a glycosylphosphatidyl inositol moiety. Since the anchor is susceptible to a glycan-specific phosphodiesterase, the pathogen, through the action of this enzyme, may rapidly shed its VSG and thus elude the immune defenses of the host. Studies on the mechanism of the immune response to carbohydrate antigens and the antigen-antibody reaction were also reported. By sequencing variable regions of light and heavy chains of 3 different monoclonal antibodies directed against the 3-fucosyllactosamine determinant, it was shown that similar immunoglobulin genes were in each case operative. There was also some sharing of idiotypic determinants among the 3 antibodies as well as other antibodies to structurally unrelated polysaccharides. Other authors presented data on the most probable binding areas of monoclonal antibodies on cancerassociated antigens, including sialy~ated Le a. Regulation of the immune response at a cellular level was the subject of several communications. Natural killer cells boosted by interferon but not interleukin-2 were shown to recognize N-glycan structures at the surface of target cells. Another group has shown that the T lymphocytes of cancer patients have more sialic acid residues at the surface than their counterparts from healthy individuals; moreover, the sialic acid of T lymphocytes from cancer patients was extensively O-acetylated. Lectins may play a role in the immunity aga~.ast diseases in several ways. Among others, they probably provide attachment sites for wandering lympho-

cytes within lymphoid organs or may aid in linking phagocytic cells to targets cells. The second area of research was well represented in the symposium. Thus it was shown that the D-galactose binding receptor of rat macrophages recognizes clusters of galactose residues at the surface of target cells rather than their anomeric linkages. Another group presented further evidence for participation of bacterial lectins in bacteria-macrophage/neutrophil adherence in vitro. Evidence was presented that lectin-neoplastic cell interaction may play a role in cancer metastasis at least in the liver. Plants deploy a different defensive strategy against pathogenic bacteria and fungi than vertebrates among others, responding with production of phytoalexins which are broad specificity antibiotics. Structures of endogenous and exogenous elicitors of phytoalexins were presented. Both types are oligosaccharides in character and act synergistically activating a variety of defensive responses. These biologically active oligosaccharides were given the name 'oligosaccharins'. Summarizing, there has been a steady progress in most areas of research on immunochemistry of giycoconjugates, though obviously I could not cite all contributions. Prospects on the immediate future of this research are exciting. Gene-encoding enzymes involved in glycoconjugate metabolism and most glycoconjugates of interest will be cloned and their primary structures deduced from nucleotide sequences of respective DNAs. The potential of thi~ experimental approach cannot be overestimated and in fact has been amply demonstrated in several papers presented at this symposium and to which I referred. Beneficiaries will be all areas of glycoconjugate research including their medical applications. In the blood group field, sequencing of genes coding for different a-2-L, a-3-L-, and a-4-L-fucosyltransferases will give a direct evidence as to the mechanism of variable expression of H and Lewis antigens in cells and secretions. Sequencing of A B O genes and their respective protein products will solve evolution of the ABO blood group system and the status of the O gene. The feasibility of 'cancer vaccines' is difficult to assess at present. It is quite possible, however, that methods of stimulation of immune response to cancer-associated antigens will find their way into routine cancer ~therapy. As far as regulation of the immune response at cellular level is concerned, I hope that the exact function of gangliosides as putative immunomodulators and cell growth regulators will be elucidated. Long-rate predictions as to future directions of glycoconjugate research are difficukt and have to be discussed in the context of the biological function of carbohydrates. For a long time an informative function for carbohydrates was postulated on the assumption that, since glycans are invariably present at the cell membrane, they must have a role in membrane-mediated information. Indeed, carbohydrates are recognized by cells of the immune system. They are recognition molecules for bacterial

1705 lectins. Endogenous animal lectins or carbohydratespecific receptors are known to be involved in a variety of processes, including intracellulaf sorting of iysosomal enzymes, cell adhesion and probably embryonic development and organ formation. Yet we should not expect too much, because the array of carbohydrate structures available to cells is in fact limited and the specificity of lectins rather broad. Some membrane glycoconjugates, as for instance glycosphingolipids of erythrocytes, are, in my opinion, membrane-packing substances which cover the membrane with a lay.er of informationally inert carbohydrate. Such inert carbohydrate may also cover protease sensitive or immunogenic sites on functional glycoproteins. We should distinguish between

primary and secondary functions of biological molecules. Nucleic acids are carriers of genetic information yet their subunits in a secondary capacity function as energy-rich compounds or sugar donors. Proteins are catalytic and information substances but may be used also as structural elements and food. The primary role of lipids is to provide the basic structure of biological membranes. In their seconry role, they are used as food and for information purposes. The primary function of carbohydrate is to provide energy and structure but they are also molecules of information. Depending upon how important this secondary function of carbohydrates in biology is, so will be the future of glycoconjugate research.

Summary and future trends of research on glycoconjugate interactions: molecular and cellular aspects Roland S C H A U E R

Biochemisches Institut, Christian-Albrechts-Universitiit, Olshausenstr. 40, D-2300 Kid, F.R.G. Among the many different functions of carbohydrates, their role as ligands for sugar-recognizing receptors (lectins), which facilitate molecular and cellular contacts, is notable. During this symposium, the participants were witness to many reports on the progress made in this field and about 140 lectures and posters gave ample opportunity for discussion. We now know much more about lectins of plant origin, however, more and more is being discovered about bacterial and mammalian lectins. Of greatest interest is the occurrence of lectins in the outer and inner membranes of mammalian cells, as well as their mobility, turnover and function. Although the carbohydrate specificity of these receptors is high with regard to the nature and the localization of sugars in oligosaccharide chains, it is not as prominent as that of carbohydrate-specific antibodies. Several of the presentations have provided new insight into the three-dimensional mode of interaction between sugars and lectins. More is now known about the primary structure of lectins, mainly because of advances in gene cloning techniques. Such investigations, will be of greater importance in the future, for the comparison of the primary structures and the sugar-binding sites of lectins with those of other sugar-recognizing proteins, such as glycosidases and glycosyltransferases. In addition, the study of genetically related lectin families in mammalian cells will also be prominent. Correspondingly, the evolution, genetic control, and relationship between leetins and the immune system are topics of interest. More

researchers will be focussing their studies on the cellular and humoral stimuli leading to lectin expression, a process which is very important in mammalian cells during development or malignancy and in specialized cells, such as macrophages. Regulation may also occur by modification of the affinity of lectins or their sialic acids, as was observed with other receptors. More data are now available about the biological and pathophysiological role of lectins. For example, how they facilitate or hinder infection by microorganisms, participate in fertilization processes, embryogenesis, tissue formation, growth inhibition caused by cell contact, differentiation of lymphocytes and erythrocytes and how they may be involved in the trapping of aged or transformed cells by macrophages, or in the formation of tumor metastases. Lectins are responsible for the binding of aggregating substances such as proteoglycans, growth-regulating compounds and several adhesins. In this connection the discovery of polysialylated glycoprotein'~ is worthy of note. These unusual molecules control neuronal development and also occur in other embryonic tissues anO in some tumors. Binding of molecules and cells to each other or to a substratum may be regulated by reversible sialylation of surface oligosaccharides, leading to masking of, for example, galactose residues, which seem to be the most frequent ligands for mammalian lectins. In this model, sialylation results in the dissociation of cells or molecules and, after desialylation, the cells