Carbohydrate analysis.

The analysis of the carbohydrate chains attached to proteins is becoming increasingly important as appreciation of the role of glycosylation in the structural and functional properties of biologically significant glycoproteins grows. Over the past year, a number of developments have been made that may improve and promote the analysis of the glycosylation of proteins.     

Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.     

Glycobiology of the neuromuscular junction

Most molecules that are present at synapses are glycosylated with carbohydrates, and some carbohydrate structures are themselves uniquely synaptic in their localization. Thus, proteins or lipids at the synapse may bear distinct carbohydrates that alter their localization or function. Here, I will review the evidence that there are unique synaptic carbohydrates at the neuromuscular junction. Then, I will review the evidence that such carbohydrates can affect the function of synaptic proteins, with particular attention to agrin, dystroglycan, and the neural cell adhesion molecule (NCAM). Finally, I will review recent data that demonstrates a role for one carbohydrate structure, the cytotoxic T cell (CT) antigen, in neuromuscular development. These studies suggest that glycosylation is an important modification to consider in studies of synapse formation and function.     

Infrared spectroscopy used to evaluate glycosylation of proteins

Infrared (IR) spectroscopy is used for studying the carbohydrate moieties of glycosylated proteins. IR spectra of mono- and disaccharides in the fingerprint region are specific to each sugar and to the environment of the sugar molecules (i.e., aqueous solution or anhydrous glass phase). The IR spectra of glycosylated proteins (mucin, soybean peroxidase, collagen IV, and avidin) were compared with those of the constituent sugars and cytochrome c (a protein with no glycosylation). Our results demonstrate that the IR absorption spectra of glycosylated proteins show distinct absorption bands for the sugar moiety, the protein amide group, and water. Therefore, IR can be used to detect glycosylation.     

Dissecting glycoprotein biosynthesis by the use of specific inhibitors

It is possible to interfere with different steps in the dolichol pathway of protein glycosylation and in the processing of asparagine-linked oligosaccharides. Thus some clues about the role of protein-bound carbohydrate can be obtained by comparing the biochemical fates and functions of glycosylated proteins with their non-glycosylated counterparts, or with proteins exhibiting differences in the type of oligosaccharide side chains. Cells infected with enveloped viruses are good systems for studying both aspects of protein glycosylation, since they contain a limited number of different glycoproteins, often with well-defined functions. Tunicamycin, an antibiotic, as well as several sugar analogues have been found to act as inhibitors of protein glycosylation by virtue of their anti-viral properties. They interfere with various steps in the dolichol pathway resulting in a lack of functional lipid-linked oligosaccharide precursors. Compounds that interfere with oligosaccharide trimming represent a second generation of inhibitors of glycosylation. They are glycosidase inhibitors that interfere with the processing glucosidases and mannosidases and, as a result, the conversion of high-mannose into complex-type oligosaccharides is blocked. Depending upon the compound used, glycoproteins contain glucosylated-high-mannose, high-mannose or hybrid oligosaccharide structures instead of complex ones. The biological consequences of the alterations caused by the inhibitors are manifold: increased susceptibility to proteases, improper protein processing and misfolding of polypeptide chains, loss of biological activity and alteration of the site of virus-budding, to name but a few.     

Carbohydrate-carbohydrate interactions in adhesion

Cell-cell interactions play an important role in the development, maintenance, and pathogenesis of tissues. They are highly dynamic processes which include migration, recognition, signaling, adhesion, and finally attachment. Cells on their pathway to a final location have to pass and interact with their substratum formed of matrix and cell layers. Testing and recognition are important keys for the proper result of tissue formation. They can, however, also lead to diseases when they are misused in pathological situations, by microorganisms or malignant cells, for instance. Carbohydrates, which are the most prominent surface-exposed structures, must play an important role as recognition molecules in such processes. The rich variability of carbohydrate sequences which cell surfaces can present to lectins, adhesion molecules, and other ligands creates a refined pattern of potential attachment sites. The subtle control of the surface presentation density can provide variations in attachment strength. Not only the carbohydrate sequences but also the fact that carbohydrates can be branched while proteins cannot and that the oligosaccharide chains can be attached to the protein backbone in different densities and patterns will create yet more interaction possibilities. Maximal use of the combinatorial richness of carbohydrate molecules would be made when carbohydrate sequences could interact with other carbohydrate sequences. Such interactions have only very rarely been considered for biochemically and biologically relevant situations since they are difficult to measure. A few are known and will be summarized here with the hope that this wealth of possible chemical interactions may be considered more and more by surface cell biochemists when analyzing fine tuning in cellular interactions. © 1996 Wiley-Liss, Inc.     

O-linked protein glycosylation structure and function

There has been a recent resurgence of interest in the post-translational modification of serine and threonine hydroxyl groups by glycosylation, because the resulting O-linked oligosaccharide chains tend to be clustered over short stretches of peptide and hence they can present multivalent carbohydrate antigenic or functional determinants for antibody recognition, mammalian cell adhesion and microorganism binding. Co-operativity can greatly increase the affinity of interactions with antibodies or carbohydrate binding proteins. Thus, in addition to their known importance in bearing tumour associated antigens in the gastrointestinal and respiratory tracts, glycoproteins with O-linked chains have been implicated as ligands or co-receptors for selectins (mammalian carbohydrate binding proteins). Microorganisms may have adopted similar mechanisms for interactions with mammalian cells in infection, by having relatively low affinity ligands (adhesins) for carbohydrate binding, which may bind with higher affinity due to the multivalency of the host ligand and which are complemented by other virulence factors such as interactions with integrin-type molecules. In addition to specific adhesion signals from O-linked carbohydrate chains, multivalent O-glycosylation is involved in determining protein conformation and forming conjugate oligosaccharide-protein antigenic, and possible functional determinants.     

Comparative properties of human alpha-1-proteinase inhibitor glycosylation variants

Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.     

Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function

The glycoprotein hormone erythropoietin (Ep), the primary regulator of erythropoiesis, is synthesized by the kidney and secreted as the mature protein with three N-linked and one O-linked oligosaccharide chains. To investigate the role(s) of each carbohydrate moiety in the biosynthesis and function of Ep, we have used oligonucleotide-directed mutagenesis of a cDNA for human Ep to alter the amino acids at each of the carbohydrate attachment sites. Each mutated cDNA construct was expressed in stably transfected sublines of a kidney cell line, baby hamster kidney. We show, by preventing attachment of N-linked carbohydrate at asparagines 38 or 83, or preventing O-linked glycosylation at serine 126, that glycosylation of each of these specific sites is critical for proper biosynthesis and secretion of Ep. Fractionation of cellular extracts demonstrated that the mutant proteins lacking glycosylation at each of these three sites, (38, 83, and 126) were associated mainly with membrane components or were degraded rapidly. Less than 10% of these three mutant proteins were processed properly and secreted from the cells. The Ep protein lacking N-linked glycosylation at asparagine 24 is synthesized and secreted as efficiently as native Ep. The carbohydrates at positions 24 and 38 may be involved in the biological activity of Ep, since the absence of either of the oligosaccharide side chains at these positions reduced the hormone's biological activity.     

Effect of Carbohydrate Position on Lysosomal Transport of Procathepsin L

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30–50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH₄Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.     

Human polyreactive and monoreactive antibodies: effect of glycosylation antigen binding

The present experiments were initiated to determine whetherthe carbohydrate portions of antibody molecules contribute topolyreactivity. Cell lines making human monoclonal polyreactiveor monoreactive antibodies of the immunoglobulin (Ig) M, IgGand IgA isotypes were treated with tunicamycin to block N-linkedglycosylation of the proteins. Analysis of the secreted nativeand non-glycosylated proteins revealed a >95% inhibitionof [³H]mannose incorporation. Electrophoresis on sodium dodecylsulphate-polyacrylamide gels of the proteins from tunicamycin-treatedcells showed increased mobility and the absence of [³H]mannoseincorporation of the immunoglobulin heavy chains, consistentwith the lack of glycosylation. The native and non-glycosylatedantibodies were then tested for their ability to bind differentantigens. Despite the lack of glycosylation, both polyreactiveand monoreactive antibodies bound to antigens with little ifany loss of reactivity or specificity. It is concluded thatthe carbohydrate moieties do not contribute significantly topolyreactivity. antigen binding glycosylation polyreactive antibodies     

Current ideas on the significance of protein glycosylation

Carbohydrate has been removed from a number of glycoproteins without major effect on the structure or enzyme activity of the protein. Thus carbohydrate has been suggested to underly a non-primary function for proteins, such as in relatively non-specific interactions with other carbohydrates or macromolecules, stabilization of protein conformation, or protection from proteolysis. This non-specific concept is consistent with both the general similarity in carbohydrate structure on very diverse glycoproteins and the frequent structural microheterogeneity of carbohydrate chains at given sites. The concept is supported in a general sense by the viability of cells whose glycosylation processes have been globally disrupted by mutation or pharmacological inhibitors.In contrast to the above observations, other studies have revealed the existence of specific, selective receptors for discrete oligosaccharide structures on glycoproteins which seem to be important for compartmentalization of the glycoprotein, or the positioning of cells on which the glycoprotein is concentrated. Sometimes multivalency in the carbohydrate-receptor interaction is crucial. There are additional possible roles for carbohydrate in the transduction of information upon binding to a receptor. The possibility of specific roles for carbohydrate is supported by the existence of numerous unique carbohydrate structures, many of which have been detected as glycoantigens by monoclonal antibodies, with unique distributions in developing and differentiated cells.This article attempts to summarize and rationalize the contradictory results. It appears that in general carbohydrate does in fact underlie only roles secondary to a protein's primary function. These secondary roles are simple non-specific ones of protection and stabilization, but often also satisfy the more sophisticated needs of spatial position control and compartmentalization in multicellular eukaryotic organisms. It is suggested that there are advantages, evolutionarily speaking, for the shared use of carbohydrate for non-specific roles and for specific roles primarily as luxury functions to be executed during the processes of cell differentiation and morphogenesis.     

Affinity enhancement by multivalent lectin–carbohydrate interaction

The binding of simple carbohydrate ligands by proteins often requires affinity enhancement to attain biologically relevant strength. This is especially true for endocytotic receptors and the molecules that engage in the first-line of defense. For such purposes, nature often utilizes a mode of affinity enhancement that arises from multiple interactions between the binding proteins and the carbohydrate ligands, which we term glycoside cluster effect. In this review article we give a number of examples and describe important factors in the multi-valent interactions that govern the degree of affinity enhancement.     

Sweet new world: glycoproteins in bacterial pathogens

In eukaryotes, the combinatorial potential of carbohydrates is used for the modulation of protein function. However, despite the wealth of cell wall and surface-associated carbohydrates and glycoconjugates, the accepted dogma has been that prokaryotes are not able to glycosylate proteins. This has now changed and protein glycosylation in prokaryotes is an accepted fact. Intriguingly, in Gram-negative bacteria most glycoproteins are associated with virulence factors of medically significant pathogens. Also, important steps in pathogenesis have been linked to the glycan substitution of surface proteins, indicating that the glycosylation of bacterial proteins might serve specific functions in infection and pathogenesis and interfere with inflammatory immune responses. Therefore, the carbohydrate modifications and glycosylation pathways of bacterial proteins will become new targets for therapeutic and prophylactic measures. Here we discuss recent findings on the structure, genetics and function of glycoproteins of medically important bacteria and potential applications of bacterial glycosylation systems for the generation of novel glycoconjugates.     

Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin.

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.     

Ethanol impairs monosaccharide uptake and glycosylation in cultured rat astrocytes

Astrocyte and glial-neuron interactions have a critical role in brain development, which is partially mediated by glycoproteins, including adhesion molecules and growth factors. Ethanol affects the synthesis, intracellular transport, subcellular distribution and secretion of these glycoproteins, suggesting alterations in glycosylation. We analyzed the effect of long-term exposure to low doses of ethanol (30 mm) on glycosylation process in growing cultured astrocytes in vitro. Cells were incubated for short (5 min) and long (90 min) periods with several radioactively labeled carbohydrate precursors. The uptake, kinetics and metabolism of these precursors, as well as the radioactivity distribution in protein gels were analyzed. The levels of GLUT1 and mannosidase II were also determined. Ethanol increased the uptake of monosaccharides and the protein levels of GLUT1 but decreased those of mannosidase II. It altered the carbohydrate moiety of proteins and increased cell surface glycoproteins containing terminal non-reduced mannose. These results indicate that ethanol impairs glycosylation in rat astrocytes, thus disrupting brain development.     

Effect of glycosylation on the mechanism of renaturation of invertase from yeast

N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.     

The membrane receptors guanylyl cyclase-A and -B undergo distinctive changes in post-translational modification during brain development

Temporal carbohydrate expression patterns at cell surfaces are thought to be of crucial regulatory significance during developmental processes. Hitherto, however, data on individual membrane proteins undergoing development-associated changes in glycosylation are sparsely. Here, we show that the two natriuretic peptide receptors, guanylyl cyclase-A (GC-A) and GC-B are subject to pronounced size alterations in the rat brain between postnatal day 1 and adult. Comparable size changes were not detectable for GC-A and GC-B in peripheral tissues and for three other membrane proteins (insulin receptor, insulin-like growth factor-II/mannose-6-phoshate receptor, neutral endopeptidase) in brain, indicating remarkable specificity. As revealed by treatments with carbohydrate-digesting enzymes, both GC-A and GC-B are hyperglycosylated at N-linked glycosylation sites in the developing brain. At postnatal day 1, the vast majority of GC-B (but not GC-A) molecules contain additionally an O-linked carbohydrate modification of about 1 kDa in mass and a further modification of similar size which is resistant to enzymatic removal. The glycoforms exhibited functional activity in membrane GC assays, indicating proper folding and signaling capability. These data link recently reported roles of natriuretic peptides during brain development for the first time with specific glycosylation states of their cyclic GMP-generating receptors.     

Mummy encodes an UDP-N-acetylglucosamine-dipohosphorylase and is required during Drosophila dorsal closure and nervous system development

Throughout development cell-cell interactions are of pivotal importance. Cells bind to each other or share information via secreted signaling molecules. To a large degree, these processes are modulated by post-translational modifications of membrane proteins. Glycan-chains are frequently added to membrane proteins and assist their exact function at the cell surface. In addition, the glycosylation pathway is required to generate GPI-linkage in the endoplasmatic reticulum. Here, we describe the analysis of the cabrio/mummy gene, which encodes an UDP-N-acetylglucosamine diphosphorylase. This is a well-conserved and central enzyme in the glycosylation pathway. As expected from this central role in glycosylation, cabrio/mummy mutants show many phenotypic traits ranging from CNS fasciculation defects to defects in dorsal closure and eye development. These phenotypes correlate well with specific glycosylation and GPI-anchorage defects in mummy mutants.     

The carbohydrate recognition domain of collectins

Collectins are effector molecules of the innate immune system that play an important role in the first line of defence against bacteria, viruses and fungi. Most of their interactions with microorganisms are mediated through their carbohydrate recognition domain (CRD), which binds in a Ca(2+)-dependent manner to glycoconjugates. This domain is a well-known structure that is present in a larger group of proteins comprising the C-type lectin domain family. Collectins form a subgroup within this family based on the presence of a collagen domain and the trimerization of CRDs, which are essential for the ligand-binding properties of these proteins. The ligand specificity among the nine collectin members is significantly different as a result of both the structural organization of the trimers and specific sequence changes in the binding pocket of the CRD. In addition, some collectin members have additional features, such as N-linked glycosylation of CRD residues and additional loop structures within the CRD that have a large impact on their interaction with the glycoconjugates present on microorganisms or host cells. The availability of crystal structures of three members of the collectin family (surfactant proteins A and D and mannan-binding protein) provides an important tool for addressing the impact of these CRD differences on ligand binding. In this review, the structural differences and similarities between the CRDs of collectins are summarized and their relationship with their ligand-binding characteristics is discussed.