Sequence determination for N-linked oligosaccharides through the use of the nuclear Overhauser effect

A nuclear magnetic resonance technique, the nuclear Overhauser effect, has been used to confirm the presence of Man alpha 1-2, GlcNAc beta 1-2, and Man alpha 1-3 residues and to establish the branching pattern and sequences in four classes of N-linked oligosaccharides. This method offers a rapid and nondestructive approach to the elucidation of sequences in carbohydrate chains, in contrast to enzymatic and chemical methods.     

NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]     

Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules.

To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site.     

Inhibitors of the biosynthesis and processing of N-linked oligosaccharides

A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Structure of the phosphorylated N-linked oligosaccharides from the mnn9 and mnn10 mutants of Saccharomyces cerevisiae

The N-linked oligosaccharides, from Saccharomyces cerevisiae mnn1 mnn9 mutant mannoprotein extracted from the cells in hot citrate buffer, were separated by ion exchange into a monophosphate diester, a monophosphate monoester, a diphosphate diester, and a diphosphate monoester diester. The structures of the major components with diesterified phosphate were assigned as follows (where M = mannose), according to a recently revised oligosaccharide structure for the mnn mutants (Hernandez, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). formula; see text The monoester derivatives were mixtures of the possible isomers produced by removal of one or the other phosphoglycosyl-linked mannose units, and they were shown to arise by chemical degradation during isolation. The mnn1 mnn2 mnn10 acidic oligosaccharide fraction contained a mono- and a diphosphate ester. The monophosphate consisted predominantly of a single isomer with a mannosyl phosphate unit located at the end of the outer chain in an oligosaccharide with the following structure, where x may range from 2 to 12. The diphosphate had a mannosyl phosphate in this formula; see text position as well as one on the terminal alpha 1----6-linked mannose in the core. The presence in the mnn1 mnn9 or mnn1 mnn2 mnn10 background of the mnn4 or mnn6 mutations, which are known to regulate phosphorylation in yeast, reduced phosphorylation by 90% but did not eliminate it. AI-12522     

HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation

Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples.     

Heterogeneity of the complex N-linked oligosaccharides at specific glycosylation sites of two secreted carrot glycoproteins

The N-linked glycans from the 52/54-kDa medium protein and cell wall beta-fructosidase, two glycoproteins secreted by carrot suspension culture cells, were characterized. Carrot cells were labelled with [3H]glucosamine or [3H]fucose. The 52/54-kDa medium protein was isolated from the culture medium and beta-fructosidase from cell walls. The purified proteins were digested with trypsin and glycopeptides were isolated and sequenced. Glycans obtained from individual glycopeptides were separated by gel filtration chromatography and characterized by concanavalin A chromatography, by treatments with exoglycosidases and by sugar composition analysis. The 52/54-kDa medium protein and cell wall beta-fructosidase have one high-mannose-type glycan similar to those from yeast and animal glycoproteins. In addition, the 52/54-kDa medium protein has three complex-type and cell wall beta-fructosidase two complex-type glycans per polypeptide. The complex-type glycans isolated from individual glycosylation sites are fairly large and very heterogeneous. The smallest of these glycans has the structure [Xyl](Man)3[Fuc](GlcNAc]2Asn (square brackets indicating branching) whereas the larger ones carry additional sugars like terminal N-acetylglucosamine and possibly rhamnose and arabinose in the case of the 52/54-kDa medium protein and only arabinose in the case of cell wall beta-fructosidase. These terminal sugars are linked to the alpha-mannose residues of the glycan cores. The 52/54-kDa medium protein is secreted with large and homogeneous complex glycans, their heterogeneity originates from slow processing after secretion. The complex glycans from cell wall beta-fructosidase are processed before the enzyme is integrated into the cell wall.     

Structural characterization of the N-linked oligosaccharides from tomato fruit.

The primary structures of the N-linked oligosaccharides from tomato fruit (Lycopersicon esculentum) have been elucidated. For the isolation of the protein fraction, two procedures were employed alternatively: a low temperature acetone powder method and ammonium sulfate precipitation of the tomato extract. After peptic digestion, the glycopeptides were purified by cation-exchange chromatography; the oligosaccharides were released by N-glycosidase A and fluorescently labelled with 2-aminopyridine. Structural characterization was accomplished by means of two-dimensional HPLC in combination with exoglycosidase digestions and MALDI-TOF mass spectrometry. Two varieties as well as two stages of ripening were investigated. In all the samples, the same sixteen N-glycosidic structures were detected; the two most abundant glycans showed identical properties to those of the major N-linked oligosaccharides of horseradish peroxidase and pineapple stem bromelain, respectively and accounted for about 65-78% of the total glycan amount; oligomannosidic glycans occurred only in small quantities (3-9%). The majority of the N-glycans were beta 1,2-xylosylated and carried an alpha 1,3-fucose residue linked to the terminal N-acetylglucosamine. This structural element contributes to cross-reactions among non-related glycoproteins and has been shown to be an IgE-reactive determinant (Tretter, Altmann, Kubelka, M?rz, & Becker, 1993). The presented study gives a possible structural explanation for reported immunological cross-reactivities between tomato and grass pollen extracts due to carbohydrate IgE epitopes (Petersen, Vieths, Aulepp, Schlaak, & Becker, 1996), thereby demonstrating the importance of the structural characterization of plant N-glycans for a more reliable interpretation of immunological data.     

Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells

The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1 Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3 Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that alpha(1-->3) fucosylation of N-glycans does occur but beta(1-->4) galactosylation of the sugar chains does not in the tobacco cultured cells.     

Folding mechanism of the CH2 antibody domain

The immunoglobulin C(H)2 domain is a simple model system suitable for the study of the folding of all-beta-proteins. Its structure consists of two beta-sheets forming a greek-key beta-barrel, which is stabilized by an internal disulfide bridge located in the hydrophobic core. Crystal structures of various antibodies suggest that the C(H)2 domains of the two heavy chains interact with their sugar moieties and form a homodimer. Here, we show that the isolated, unglycosylated C(H)2 domain is a monomeric protein. Equilibrium unfolding was a two-state process, and the conformational stability is remarkably low compared to other antibody domains. Folding kinetics of C(H)2 were found to consist of several phases. The reactions could be mapped to three parallel pathways, two of which are generated by prolyl isomerizations in the unfolded state. The slowest folding reaction, which was observed only after long-term denaturation, could be catalyzed by a prolyl isomerase. The majority of the unfolded molecules, however, folded more rapidly, on a time-scale of minutes. Presumably, these molecules also have to undergo prolyl isomerization before reaching the native state. In addition, we detected a small number of fast-folding molecules in which all proline residues appear to be in the correct conformation. On both prolyl isomerization limited pathways, the formation of partly structured intermediates could be observed.     

N-linked oligosaccharides affect the enzymatic activity of CD39: diverse interactions between seven N-linked glycosylation sites

Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, has seven potential N-glycosylation sites at asparagine residues 73, 226, 291, 333, 375, 429, and 458. To determine their roles in the structure and function of CD39, we mutated these sites individually or in combination by replacing asparagine with serine or glutamine and analyzed the surface expression and the enzymatic activity of the mutants. The results indicate that rat CD39 can be glycosylated at all seven sites when expressed in COS7 cells. Glycosylation sites 73 at the N terminus, 333 in the middle, and 429 and 458 at the C terminus were principally required for cell surface appearance of enzymatically active CD39. Whereas deletion of these sites individually had modest effects on surface ATPase activity, some double deletions of these sites had major effects on both surface activity and expression. The importance of these N-glycosylation sites is recognizable in other members of the ectonucleoside triphosphate diphosphohydrolase family.     

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides

We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648-13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. (see structure below)     

The reduced form of the antibody CH2 domain

Among the immunoglobulin domains, the CH2 domain has the lowest thermal stability, which also depends on amino acid sequence and buffer conditions. To further identify factors that influence CH2 folding and stability, we characterized the domain in the reduced form using differential scanning fluorimetry and nuclear magnetic resonance. We show that the CH2 domain can fold, similarly to the disulfide‐bridged form, without forming a disulfide‐bridge, even though the protein contains two Cys residues. Although the reduced form exhibits thermal stability more than 15°C lower than the disulfide‐bridged form, it does not undergo immediate full oxidization. To explain this phenomenon, we compared CH2 oxidization at different conditions and demonstrate a need for significant fluctuation of the folded conformation to enhance CH2 disulfide‐bridge formation. We conclude that, since CH2 can be purified as a folded, semi‐stable, reduced protein that can coexist with the oxidized form, verification of the level of oxidization at each step is critical in CH2 engineering studies.     

Pharmacokinetics of engineered human monomeric and dimeric CH2 domains

Therapeutic monoclonal antibodies have several advantages over small molecule drugs and small proteins and peptides, including a long serum half-life. The long serum half-life of IgG is due, in part, to its molecular weight (150kDa) and its ability to bind FcRn. Both the CH2 and CH3 domains of Fc are involved in FcRn binding. Antibody fragments and antibody-like scaffolds have improved penetration into tissues due to their small size, yet suffer from a short serum half-life of less than one hour. The human CH2 domain (CH2D) of IgG1 retains a portion of the FcRn binding site, is amenable to modification for target binding, and may represent the smallest antibody-like scaffold retaining a relatively long serum half-life. Here we describe the generation of a dimeric CH2D (dCH2D) and determination of its pharmacokinetics (PK), as well as the PK of wild-type monomeric CH2D (mCH2D) and a short stabilized CH2D variant (ssCH2D) in normal B6 mice, human FcRn transgenic mice and cynomolgus macaques. The elimination half-life of dCH2D was 9.9, 10.4 and 11.2 hours, and that of ssCH2D was 13.1, 9.9 and 11.4 hours, in B6 mice, hFcRn mice and cynomolgus macaques, respectively. These half-lives were slightly longer than that of mCH2D (6.9 and 8.8 hours) in B6 and hFcRn mice, respectively. These data demonstrate that engineered CH2D-based variants have relatively long serum half-lives, making them a unique scaffold suitable for development of targeted therapeutics.     

Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction

The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process.     

Pharmacokinetics of engineered human monomeric and dimeric CH2 domains

Therapeutic monoclonal antibodies have several advantages over small molecule drugs and small proteins and peptides, including a long serum half-life. The long serum half-life of IgG is due, in part, to its molecular weight (150kDa) and its ability to bind FcRn. Both the CH2 and CH3 domains of Fc are involved in FcRn binding. Antibody fragments and antibody-like scaffolds have improved penetration into tissues due to their small size, yet suffer from a short serum half-life of less than one hour. The human CH2 domain (CH2D) of IgG1 retains a portion of the FcRn binding site, is amenable to modification for target binding, and may represent the smallest antibody-like scaffold retaining a relatively long serum half-life. Here we describe the generation of a dimeric CH2D (dCH2D) and determination of its pharmacokinetics (PK), as well as the PK of wild-type monomeric CH2D (mCH2D) and a short stabilized CH2D variant (ssCH2D) in normal B6 mice, human FcRn transgenic mice and cynomolgus macaques. The elimination half-life of dCH2D was 9.9, 10.4 and 11.2 hours, and that of ssCH2D was 13.1, 9.9 and 11.4 hours, in B6 mice, hFcRn mice and cynomolgus macaques, respectively. These half-lives were slightly longer than that of mCH2D (6.9 and 8.8 hours) in B6 and hFcRn mice, respectively. These data demonstrate that engineered CH2D-based variants have relatively long serum half-lives, making them a unique scaffold suitable for development of targeted therapeutics.     

Porcine pulmonary collectins show distinct interactions with influenza A viruses: role of the N-linked oligosaccharides in the carbohydrate recognition domain

Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals. Pigs can serve as important intermediate hosts for transmission of avian IAV strains to humans, and for the generation of reassortant strains; this may result in the appearance of new pandemic IAV strains in humans. We have studied the role of the porcine lung collectins surfactant proteins D and A (pSP-D and pSP-A), two important components of the innate immune response against IAV. Hemagglutination inhibition assays revealed that both pSP-D and pSP-A display substantially greater inhibitory activity against IAV strains isolated from human, swine, and horse, than lung collectins from other animal species. The more potent activity of pSP-D results from interactions mediated by the asparagine-linked oligosaccharide located in the carbohydrate recognition domain of pSP-D, which is absent in SP-Ds from other species characterized to date. Presence of this sialylated oligosaccharide moiety enhances the anti-influenza activity of pSP-D, as demonstrated by assays of viral aggregation, inhibition of infectivity, and neutrophil response to IAV. The greater hemagglutination inhibitory activity of pSP-A is due to porcine-specific structural features of the conserved asparagine-linked oligosaccharide in the carbohydrate recognition domain of SP-A. A more efficient lung collectin-mediated immune response against IAV in pigs may play a role in providing conditions by which pigs can act as "mixing vessel" hosts that can lead to the production of reassortant, pandemic strains of IAV.     

A mathematical model of sialylation of N-linked oligosaccharides in the trans-Golgi network

A mathematical model is developed of the compartmentalized sialylationof N-linked oligosaccharides in order to understand and predictthe outcome of sialylation reactions. A set of assumptions arepresented, including Michaelis-Menten-type dependency of reactionrate on the concentration of the glycoprotein substrate. Theresulting model predicts the heterogeneous outcome of a posttranslationaloligosaccharide biosynthesis step, a critical aspect that isnot accounted for in the modeling of the cotranslational attachmentof oligosaccharides to glycosylation sites (Shelikoff et al.,Biotech. Bioeng., 50, 73–90, 1996) or general models ofthe secretion process (Noe and Delenick, J. Cell Sci, 92, 449–459,1989). In the steady-state for the likely case where the concentrationof substrate is much less than the K_m of the sialyltransferase,the model predicts that the extent of sialylation, x, will dependupon the enzyme concentration, enzyme kinetic parameters andsubstrate residence time in the reaction compartment. The valueof x predicted by the model using available literature datais consistent with the values of x that have been recently determinedfor the glycoproteins CD4 (Spellman et al, Biochemistry, 30,2395–2406, 1991) and t-PA (Spellman et al, J. Biol Chem.,264, 14100–14111, 1989) secreted by Chinese hamster ovarycells. For the unsaturated case, the model also predicts thatx is independent of the concentration of secreted glycoproteinin the Golgi. The general modeling approach outlined in thisarticle may be applicable to other glycosylation reactions andposttranslational modifications. model sialylation N-linked glycosylation