The hope that hybridoma antibodies would reveal unique cell surface antigens during embryogenesis, differentiation and oncogenesis has been replaced by the realization that such antigens are mainly carbohydrate structures of glycoproteins and glycolipids occurring in many cell types. These findings either may reflect limitations in the methods of selection of hybridoma antibodies or may point to important roles for the diverse carbohydrate structures as receptors for regulators of cell growth and differentiation.

Demonstration by Monoclonal Antibodies That Carbohydrate Structures of Glycoproteins and Glycolipids Are Onco-Developmental Antigens

Publisher Summary Aberrant glycosylation is the most common phenomenon associated with oncogenic transformation expressed in cell membranes of animal and human cancer cells. Such aberrant structures at the surface membranes may well be effective targets in prevention, diagnosis, and treatment of human cancer. Many of the aberrant glycosylation products can be recognized by specific MAbs as tumor-associated carbohydrate antigens. Many of these antigens have been identified as carbohydrates, thus there is increasing evidence that essentially all human cancers are characterized by aberrant glycosylation. Aberrant glycosylation as such may be the basis of inappropriate cell/cell and cell/matrix interactions that may be reflected in the abnormal cell social behavior of tumor cells, such as uncontrolled cell growth, invasiveness, and metastatic potential. Whatever the genetic or epigenetic basis of expression of aberrant glycosylation is, the phenomenon is of crucial importance in understanding the antisocial behavior of tumor cells as well as in practical applications in diagnosis and treatment of human cancer.

Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens

A current trend in cell biology is to study the mechanisms of regulatory cellular function through specific structural and func tional molecules, i.e., growth factors, their receptors, membrane transporters, cytoskeletal systems, surface membrane and pericellular components, etc., rather than classical enzymes. The key molecules involved in such functional regulation are modified by 2 major chemical processes, phosphorylation and glycosylation. A greal deal of interest has been focused on phosphorylation as a ubiquitous and essential modulatory mechanism of cellular function. This idea has been greatly reinforced by the fact that many transforming genes (cellular as well as viral oncogenes) encode phosphoproteins, which by themselves are protein kinases, although the functions of many of their substrates have not been identified (see, for a review, Ref. 9). Less attention has been paid to the functional significance of glycosylation than to that of phosphorylation; nevertheless, the changes in glycosylation occur as quickly and dramatically as the process of phosphorylation at various stages of develop ment, differentiation, and oncogenesis (see, for a review, Ref. 38). While phosphorylation patterns are few, the patterns of glycosylation are numerous; over 100 types of glycosylated structures have been characterized among those bound to lipids (ceramides), i.e., glycolipids (see, for a review, Ref. 39). Similar variation of glycosylation can also be found in the peripheral region of carbohydrates in glycoproteins, in addition to the structural variation in the core region of carbohydrate chains bound to proteins through Nor O-glycosides (see, for reviews, Refs. 42 and 65). In contrast to phosphorylation, which directly modulates pro tein function, glycosylation affects the conformation, localization, and organization of functional and structural proteins; thus, glycosylation determines the turnover and organizational frame work of proteins within cytoplasm, membranes, and the pericellular matrix (see, for reviews, Refs. 42 and 87). LJpidglycosylation in membranes, particularly cell surface membranes, offers cell recognition sites for cell-social events and may regulate the function of membrane proteins such as receptors and adhesive proteins (38, 115) (Table 1). With the functional significance of

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Aberrant glycosylation in cancer cell membranes as focused on glycolipids: overview and perspectives.

The Biochemistry of glycoproteins and proteoglycans

Publisher Summary This chapter discusses the application of high-resolution, 1H-nuclear magnetic resonance (NMR) spectroscopy to the structural analysis of carbohydrates related to glycoproteins. Glycoproteins are biopolymers consisting of a polypeptide backbone bearing one or more covalently linked carbohydrate chains. The carbohydrate chains of glycoproteins may be classified according to the type of linkage to the polypeptide backbone. N-Glycosylic chains are attached to the amide group of asparagine (Asn), whereas the O-glycosylic chains are linked to the hydroxyl group of amino acid residues such as serine (Ser), threonine (Thr), and hydroxylysine (Hyl). The high-resolution, 1H-NMR spectroscopy technique, in conjunction with methylation analysis, is extremely suitable for the structural studies of N-, as well as on O-, glycosylic glycans. For the interpretation of the 1H-NMR spectrum of a carbohydrate chain in terms of primary structural assignments, the concept of “structural reporter groups” was developed. This means that the chemical shifts of protons resonating at clearly distinguishable positions in the spectrum, together with their coupling constants and the line widths of their signals, bear the information essential to permit the assigning of the primary structure. This chapter presents literature data on the high-resolution, 1H-NMR spectroscopy of carbohydrates derived from glycoconjugates. It also discusses the results for carbohydrates related to the glycoproteins of N-glycosylic type.

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High-Resolution, 1H-Nuclear Magnetic Resonance Spectroscopy as a Tool in the Structural Analysis of Carbohydrates Related to Glycoproteins

Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.

Fractionation of glycopeptides by affinity column chromatography on concanavalin A-sepharose.

Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity.

Endo-β-N-acetylglucosaminidase Acting on Carbohydrate Moieties of Glycoproteins PURIFICATION AND PROPERTIES OF THE ENZYME FROM DIPLOCOCCUS PNEUMONIAE

From embryonal carcinoma cells labeled with fucose, two main classes of glycopeptide products of Pronase digestion can be distinguished by Sephadex G-50 column chromatography: one eluted near the excluded volume and a smaller one. The large fucosyl-glycopeptides are scarcely present in differentiated cells derived from embryonal carcinoma cells (i.e., fibroblastlike cells, myoblasts, and parietal yolk-sac carcinoma). During in vitro differentiation of embryonal carcinoma cells, these large glycopeptides disappear almost completely. The small glycopeptides were analyzed by paper electrophoresis, concanavalin A-Sepharose affinity chromatography, and digestion with an endoglycosidase. The major components of these glycopeptides from embryonal carcinoma cells appear to be different from complex glycopeptides known to occur in adult cells. The glycopeptide pattern of mouse preimplantation embryos resembles that of embryonal carcinoma cells. These results suggest that the carbohydrate profile changes fundamentally during early stages of mammalian development.

https://www.pnas.org/content/pnas/75/5/2315.full.pdf

Carbohydrate structure and cell differentitation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells

IN comparative gel filtration of glycopeptides obtained from the surface of normal and transformed cells, an increased concentration of fucosyl glycopeptides of apparently large molecular weight was observed for cells transformed by viral, chemical and spontaneous methods1–7. This is considered to be an expression of transformed phenotype, since in cells transformed by a temperature-sensitive (ts) mutant of Rous sarcoma virus, the heavy glycopeptides increased only at the permissive temperature. Based on the results of sialidase treatment, it was suggested that the presence of extra sialic acid is responsible for the higher molecular weight nature of the glycopeptides from transformed cells4–6.

Takashi Muramatsu

Papers

Fractionation of glycopeptides by affinity column chromatography on concanavalin A-sepharose.

Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity.

Endo-β-N-acetylglucosaminidase Acting on Carbohydrate Moieties of Glycoproteins PURIFICATION AND PROPERTIES OF THE ENZYME FROM DIPLOCOCCUS PNEUMONIAE

Carbohydrate structure and cell differentitation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells

New structural characteristic of the large glycopeptides from transformed cells