Most cell surface and secreted proteins are modified by the addition of N-glycosyl and O-glycosyl oligosaccharides, the functions of which, in many cases, remain unclear. The roles of protein glycosylation were first studied using enzyme inhibitors, glycosidases and cell mutants with altered sugar addition or modification enzymes. More recently, site-directed mutagenesis has allowed the targeting of specific oligosaccharides on a single protein species. Loss of N-linked oligosaccharides from glycoproteins often results in altered disulfide bonding and leads to the formation of aggregates probably resulting from an anomalous protein conformation. The misfolded proteins are frequently retained in the endoplasmic reticulum where they may be degraded. Many receptors lacking N-linked oligosaccharides fail to bind their ligands, again suggesting that they may be misfolded. Site-directed mutagenesis has demonstrated that individual sugar chains may have different functions in a glycoprotein and one sugar chain may have a dominant effect on the acquisition of the correct conformation. In some cases, once the correct conformation has been achieved, N-glycosyl chains appear to be dispensable. The role of O-glycosyl chains in glycoprotein function is less well understood and, where they have been studied, their loss seems to have little effect in proteins produced by many cultured cells. It is likely that these sugar chains play a role that is only apparent in multicellular situations.
There are two major families of carbohydrate-binding proteins (lectins) in the animal kingdom: the family of calcium-dependent (C-type) lectins and the family of metal-independent β-galactoside-binding lectins (so-called S-type). As one of the approaches to an understanding of reasons for their being, a general comparison of these protein families is attempted. C-type lectins form a vast family consisting of various extracellular proteins including both lectins and lectinrelated proteins. Although they have diverse molecular architectures and functions, insofar as they are known, each molecule shows rather limited biological functions and histological distributions. Such C-type lectins can be regarded as “bricolage products”, developed in the course of molecular evolution having made the best use of the carbohydraterecognition domain (CRD). On the other hand, S-type lectins form a relatively small family. At the moment, only three types of soluble metal-independent β-galactoside-binding lectins are members of this family. Though their biological roles remain elusive, recent findings of apparent homologues in some of the most primitive multicellular animals, such as sponge and nematode, strongly suggest their fundamental importance. Marked evidence that they are initially synthesized as cytoplasmic proteins implies so far unknown cytoplasmic function(s). They are possibly involved in the “essential minimum” functions for all multicellular animals in cooperation with their partner glycoconjugates, such as lactosaminoglycans. All of. these contrastive properties represent the S-type lectins as “antithesis” to the C-type lectins.
Implantation is a complex developmental process in which the early embryo becomes embedded in the uterine wall. A key step appears to be transformation of the uterine epithelium from a non-adhesive to an adhesive (hospitable) state under the influence of steroid hormones and cytokines. This period of uterine receptivity is known to be associated with numerous glycosylation changes, including changes in the glycolipid and glycoprotein composition of the uterine epithelium, the size and charge of the apical glycocalyx, and the profile of glycoproteins secreted into the luminal fluid. The embryo also undergoes rapid changes in carbohydrate antigen expression prior to implantation. These stage-specific changes may regulate the time and place of blastocyst attachment within the uterus. Severval testable hypotheses concerning the role of saccharides in implantation have emerged, invoking both carbohydrate-protein and carbohydrate-carbohydrate interactions.
The natural diversity in glycan structures found on glycoproteins appears to be due in large part to tissue-specific regulation of glycosyltransferase activities. In this review, we describe a method for examining tissue-specific patterns of O-glycosylation using aryl-α-D-GalNAC to prime O-glycan biosynthesis in vivo. The addition of p-nitrophenyl-, phenyl-, or benzyl-α-D-GalNAc to cells in tissue culture competitively inhibits extension of GalNAcα-Ser/Thr on endogenous glycoproteins, while priming aryl-oligosaccharide biosynthesis. The aryl-oligosaccharidesare produced in the Golgi and move by membrane bulk-flow into the culture medium. Absorbance of the aryl group at 303nm can be used to follow the purification of aryl-oligosaccharides, and sufficient glycan can be made for analysis by proton nuclear magnetic resonance and mass spectrometry. Structural analysis of oligosaccharides primed by aryl-α-D-GalNAc in vivo, combined with measurements of glycosyltransferase activity in vitro provide a means of characterizing O-glycosylation in diverse cell types. The limitations of this approach are also discussed.
Epidermal growth factor (EGF) is a small polypeptide mitogen that binds to specific receptors expressed at the surface of responsive cells. These receptors consist of a transmembrane glycoprotein with an extracellular domain that binds EGF and a cytoplasmic domain with intrinsic tyrosine kinase activity. Treatment of cells with EGF causes an increase in the receptor tyrosine kinase activity. This increased tyrosine phosphorylation initiates a cascade of signal transduction pathways that lead to cellular proliferation. The mechanism of activation of the receptor is mediated by an allosteric process that involves dimeric receptor complexes. The function of the EGF receptor is also regulated in vivo by phosphorylation at several sites by protein serine/threonine kinases that are activated by following treatment of cells with EGF. The EGF receptor is therefore an allosteric enzyme that is regulated by multi-site phosphorylation. Significantly, these allosteric and covalent regulatory mechanisms are altered in the presence of sphingolipids. Thus, endogenous sphingolipids represent putative physiological modulators of the function of the EGF receptor. In this review, the results of recent studies designed to test the hypothesis that sphingolipids regulate the signaling function of the EGF receptor are summarized.