Asparagine-linked Oligosaccharides Research Articles

Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes.

Expression of the T cell antigen receptor (TCR) on the surface of thymocytes and mature T cells is dependent on the assembly of receptor subunits into TCRs in the endoplasmic reticulum (ER) and their successful traversal of the secretory pathway to the plasma membrane. TCR subunits that fail to exit the ER for the Golgi complex are degraded by nonlysosomal processes that have been referred to as "ER degradation". The molecular basis for the loss of the TCR CD3-delta and TCR-alpha subunits from the ER was investigated in lymphocytes. For CD3-delta, we describe a process leading to its degradation that includes trimming of mannose residues from asparagine-linked (N-linked) oligosaccharides, generation of ubiquitinated membrane-bound intermediates, and proteasome-dependent removal from the ER membrane. When either mannosidase activity or the catalytic activity of proteasomes was inhibited, loss of CD3-delta was markedly curtailed and CD3-delta remained membrane bound in a complex with CD3-epsilon. TCR-alpha was also found to be degraded in a proteasome-dependent manner with ubiquitinated intermediates. However, no evidence of a role for mannosidases was found for TCR-alpha, and significant retrograde movement through the ER membrane took place even when proteasome function was inhibited. These findings provide new insights into mechanisms employed to regulate levels of TCRs, and underscore that cells use multiple mechanisms to target proteins from the ER to the cytosol for degradation.

Isomeric Differentiation of Asparagine-Linked Oligosaccharides by Matrix-Assisted Laser Desorption–Ionization Postsource Decay Time-of-Flight Mass Spectrometry

Matrix-assisted laser desorption–ionization (MALDI)–postsource decay (PSD) was used to differentiate glycoprotein-released N-linked oligosaccharide isomers directly from aliquots of glycosidase digests and peak fractions collected from high-pH anion exchange chromatography (HPAEC) with minimal sample handling and material. With the implementation of instrumental tuning and acquisition controls, MALDI–PSD of NMR-characterized high-mannose, hybrid, and complex standards resulted in spectra with reproducible fragment ion peak intensity ratios which correlated well to the respective oligosaccharide branching patterns. A “knowledge-based” strategy was utilized to characterize unknown isomericN-glycan structures in which specific fragment ion types and their distributions in the unknown PSD spectrum were compared to those in PSD spectra of standards possessing similar structural features. This PSD knowledge-based isomeric differentiation strategy was applied to distinguishing recombinant glycoprotein-derived Man7 D1 versus D2/D3 isomers directly from a PNGaseF digest aliquot of high-mannoseN-glycans based on branching differences. A precursor ion selection device was employed to isolate the component of interest from the mass profile without additional chromatographic isolation steps. MALDI–MS signal-to-background was maximized for direct PSD with on-the-probe sample clean-up methods. The asialo complexN-glycan PSD knowledge base was used to differentiate HPAEC peak fractions containing the tri- and tri′-antennary branching isomers and two tetraantennary isomers with antennal versus core fucose locations. Although the particular asialo complexN-glycan isomers here were well separated by HPAEC, MALDI–MS alerted us to their presence usingm/z-derived monosaccharide compositions and PSD fragmentation allowed us to differentiate these structures using the HPAEC elution positions as guides.

Sites of Asparagine-Linked Oligosaccharides in Porcine 32 kDa Enamelin

The 32 kDa enamelin protein isolated from developing porcine enamel was previously shown to contain eight different asparagine-linked oligosaccharides. However, only three consensus attachment sites were evident in this protein. In this study, glycopeptides containing all three potential glycosylation sites (72-Asn, 79-Asn and 91-Asn) were purified from 32 kDa enamelin. The oligosaccharides were isolated from each glycopeptide following digestion with N-oligosaccharide glycopeptidase, labeled with 2-aminopyridine at the reducing ends, and then characterized by reverse phase HPLC. All three potential sites were found to be glycosylated heterogeneously (i.e., five biantennary complexes at 72-Asn, two biantennary complexes at 79-Asn, three triantennary complexes at 91-Asn), accounting for all eight oligosaccharides characterized previously. These results indicate that 32 kDa enamelin has a complex pattern of asparagine-linked glycosylation localized within a small region (20 residues) of the protein. The functional significance of this glycosylation remains to be established.

タンパク質の精密質量決定の生物学的意義

Achievement of the unit mass accuracy in the molecular mass determination of proteins has been stressed. Single mass change of proteins is caused by single amino acid substitutions including Glu/Lys, Asn/Leu, Asn/Ile, Asp/Asn, and Glu/Gln, of which four mutations except for Asn/Leu are produced by single nucleotide changes. In the case of Hemoglobin Hoshida, a substitution of Asn for Asp was found in a peptide of 2056.9 Da with a decrease of single mass unit. In a structural study of the transferrin isoform which is characteristic of carbohydrate-deficient glycoprotein syndrome and missing asparagine-linked oligosaccharides, assignment of Asn but not Asp for the residue that should be glycosylated was necessary for delineating the molecular pathogenesis of this disorder. However, as demonstrated in β2-microglobulin, proteins are often partially deamidated during sample preparation or in vivo. This may compromise the biological significance of the precise mass measurement of proteins.

Analysis of asparagine-linked oligosaccharides by sequential lectin-affinity chromatography.

Sugar moieties on the cell surface play one of the most important roles in cellular recognition. In order to elucidate the molecular mechanism of these cellular phenomena, assessment of the structure of sugar chains is indispensable. However, it is difficult to elucidate the structures of cell surface oligosaccharides because of two technical problems. First is the difficulty in fractionating various oligosaccharides heterogeneous in the number, type, and substitution patterns of outer sugar branches. The second problem is that very limited amounts of material can be available, which makes it difficult to perform detailed structural studies. Lectins are proteins with sugar-binding activity. Each lectin binds specifically to a certain sugar sequence in oligosaccharides and glycopeptides To overcome the aforementioned problems, lectins are very useful tools. Recently, many attempts have been made to fractionate oligosaccharides and glycopeptides on immobilized lectin columns. The use of a series of immobilized lectin columns, whose sugar-binding specificities have been precisely elucidated, enables us to fractionate a very small amount of radioactive oligosaccharides or glycopeptides (∼10 ng depending on the specific activity) into structurally distinct groups. In this chapter, we summarize the serial lectin-Sepharose affinity chromatographic technique for rapid, sensitive, and specific fractionation and analysis of asparagine-linked oligosaccharides of glycoproteins.

Remodeling of glycoconjugates on CD44 enhances cell adhesion to hyaluronate, tumor growth and metastasis in B16 melanoma cells expressing beta1,4-N-acetylglucosaminyltransferase III.

Beta1-4 N-acetylglucosaminyltransferase III (GnT-III) synthesizes bisecting N-acetylglucosamine structures on asparagine-linked oligosaccharides. Using B16-hm mouse melanoma cells stably expressing GnT-III activity as positive transfectants, the effect of bisecting N-acetylglucosamine on the function of CD44 was analyzed in association with adhesion to hyaluronate and tumor spread in mice. Transfection of GnT-III caused increased affinity of immunoprecipitated CD44 to erythro-agglutinating phytohemagglutinin, that preferentially recognizes bisecting N-acetylglucosamine, without affecting the surface CD44 amount, indicating an increase in bisecting N-acetylglucosamine residues on CD44 in positive transfectants. CD44-mediated adhesion to immobilized hyaluronate and the binding of fluorescence-labeled hyaluronate to the cell surface were increased in positive transfectants. The enhanced adhesion in positive transfectants was suppressed by the treatment with beta-N-acetylhexosaminidase, indicating that N-acetylglucosamine residues were responsible for the enhanced adhesion. Positive transfectants showed promoted CD44-mediated tumor growth and metastatic development in the spleen after subcutaneous inoculation into mice. These results indicate that glycosylation of CD44 due to GnT-III causes enhanced adhesion to hyaluronate, local tumor growth and metastatic growth in spleen, suggesting that the CD44-mediated adhesion and tumor spread can be modified through introduction of a glycosyltransferase gene.

Elevations in Cathepsin B Protein Content and Enzyme Activity Occur Independently of Glycosylation during Colorectal Tumor Progression

Western blots, enzyme assays, protein glycosylation studies, and immunohistochemical staining were used to characterize cathepsin B expression at successive stages of colorectal tumor progression. In normal colon mucosa and premalignant adenomas, cathepsin B expression was predominantly due to mature two-chain protein detected on Western blots as the nonglycosylated 27-kDa form, with overexpression of this protein occurring in only 4 of 18 adenomas. Overexpression increased significantly in Dukes A and B carcinomas (26 of 37 cases), with cathepsin B protein generally detectable in carcinomas as a combination of both 27-kDa nonglycosylated and 28-kDa glycosylated mature two-chain forms. Glycosylated cathepsin B protein in carcinoma extracts was sensitive to PNGase F but resistant to Endo H, indicating a pattern consistent with complex rather than high mannose type glycosylation. When sorted by advancing tumor stage, peak expression of cathepsin B protein occurred in carcinomas involved in local invasion compared with adenomas or metastatic cancers. At all stages, cathepsin B activity correlated significantly with the levels of heavy chain mature cathepsin B protein (r = 0.6682, p < 0.0001) irrespective of glycosylation. Immunohistochemical staining of cathepsin B protein revealed fine diffuse cytoplasmic staining in both adenomas and carcinomas compared with coarse granular cytoplasmic staining (typical of lysosomes) seen in matched normal mucosa. Our results demonstrate several sequential, apparently independent changes in cathepsin B expression during colorectal tumor progression including early changes in subcellular localization, up-regulation of cathepsin B protein and activity in invasive cancers, and altered protein glycosylation detected in malignant tumors at all stages.

Carbohydrate analysis of porcine thyroglobulin isoforms with different iodine contents

To further validate the relationship between thyroid hormone formation and the carbohydrate structure of thyroglobulin (Tg), we reinvestigated the relationship between the iodine content and the asparagine-linked oligosaccharide structures of porcine Tg. Purified porcine Tg was further separated into isoforms (Tg-F1, -F2 and -F3) with a DEAE–cellulose ion-exchange chromatography column. The iodine residues, neutral sugar and sialic acid were analyzed for the separated Tg isoforms and their asparagine-linked oligosaccharide structures were analyzed. The asparagine-linked oligosaccharides were released from Tg-F1, -F2 and -F3 by hydrazinolysis and each oligosaccharide was labeled with p-aminobenzoic acid octyl ester (ABOE). The ABOE-labeled oligosaccharides from Tg-F1, -F2 and -F3 were analyzed for their relative content in oligosaccharides of each structure type by chemical methods and DEAE- and ConA high-performance liquid chromatography (HPLC) columns. As a result, it was revealed that the Tg fraction eluted at higher ionic strength from a DEAE–cellulose column is apt to contain more of each iodoamino acid, as well as total content of iodine, larger negative zeta-potential, conforming to sialic acid content in the Tg molecule and to a higher content of di-sialo-bi-antennary complex and to high mannose-type oligosaccharides. These results support the conclusion that iodine organification of the Tg molecule is correlated with asparagine-linked oligosaccharide completion.

Alpha-Mannosidase-II Deficiency Results in Dyserythropoiesis and Unveils an Alternate Pathway in Oligosaccharide Biosynthesis

Alpha-mannosidase-II (αM-II) catalyzes the first committed step in the biosynthesis of complex asparagine-linked (N-linked) oligosaccharides (N-glycans). Genetic deficiency of αM-II should abolish complex N-glycan production as reportedly does inhibition of αM-II by swainsonine. We find that mice lacking a functional αM-II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N-glycans. Unexpectedly, nonerythroid cell types continued to produce complex N-glycans by an alternate pathway comprising a distinct α-mannosidase. These studies reveal cell-type-specific variations in N-linked oligosaccharide biosynthesis and an essential role for αM-II in the formation of erythroid complex N-glycans. αM-II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II.

Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.

Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha2beta2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered alpha(II) subunit from human sources. The mRNA for the alpha(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (alpha(II))2beta2 tetramer was demonstrated in cultured human WI-38 and HT-1080 cells. The type II tetramer was found to represent about 30% of the total prolyl 4-hydroxylase in these cells and about 5-15% in various chick embryo tissues. The results of coexpression in insect cells argued strongly against the formation of a mixed alpha(I)alpha(II)beta2 tetramer. PDI/beta polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/beta polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source for a simple large scale purification of the recombinant enzyme. The properties of the purified human type II enzyme were very similar to those of the type I enzyme, but the Ki of the former for poly(L-proline) was about 200-1000 times that of the latter. In agreement with this, a minor difference, about 3-6-fold, was found between the two enzymes in the Km values for three peptide substrates. The existence of two forms of prolyl 4-hydroxylase in human cells raises the possibility that mutations in one enzyme form may not be lethal despite the central role of this enzyme in the synthesis of all collagens.

Generation and Characterization of Mouse Monoclonal Antibodies Specific for N-Linked Neutral Oligosaccharides of Glycoproteins

We generated four monoclonal antibodies (MAbs) specific for asparagine-linked neutral oligosaccharides of glycoproteins by immunizing mice with neoglycolipids, which were derived from glycoproteins by conjugation to phosphatidylethanolamine dipalmitoyl. The binding specificity of these MAbs was determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. The four MAbs designated OMB3, OMB4, OMR5, and OMR6 reacted strongly with the neoglycolipids, Galβ14GlcNAcβ1-2Manα1-6(Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAc-PD, GlcNAcβ1-2Manα1-6(GlcNAcβ12Manα1-3)(GlcNAcβ1-4)Manβ1-4GlcNAcβ1-4GlcNAcPD, Manα1-6Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc-PD, and Manα1-3Manβ1-4GlcNAc-PD, respectively, that were used as immunogens. All of these MAbs exhibited a high binding specificity. The epitopes of the MAbs OMB3 and OMB4 were suggested to be nonreducing terminal trisaccharides, Galβ1-4GlcNAcβ1-2Man-, and nonreducing β-GlcNAc residues, respectively. MAbs OMR5 and OMR6 showed a highly restricted binding specificity, reacting only with the immunizing neoglycolipids. Subsequently, MAbs OMB3 and OMB4 were shown to react strongly with asialo-α1-acid-glycoprotein and asialo-agalacto-α1-acid-glycoprotein, respectively, by Western blotting. Furthermore, it was shown that these MAbs reacted specifically with the epitope on Chinese hamster ovary cells by an immunofluorescence technique. MAb OMB4 was also shown to detect the accumulated oligosaccharides with nonreducing terminal β-GlcNAc residues as granular inclusions in the cultured fibroblasts from a classical Sandhoff disease patient.

Structure of glycan moieties responsible for the extended circulatory life time of fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase.

Cholinesterases are serine hydrolases that can potentially be used as pretreatment drugs for organophosphate toxicity, as drugs to alleviate succinylcholine-induced apnea, and as detoxification agents for environmental toxins such as heroin and cocaine. The successful application of serum-derived cholinesterases as bioscavengers stems from their relatively long residence time in the circulation. To better understand the relationship between carbohydrate structure and the stability of cholinesterases in circulation, we determined the monosaccharide composition, the distribution of various oligosaccharides, and the structure of the major asparagine-linked oligosaccharides units present in fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase. Our findings indicate that 70-80% of the oligosaccharides in both enzymes are negatively charged. This finding together with the molar ratio of galactose to sialic acid clearly suggests that the beta-galactose residues are only partially capped with sialic acid, yet they displayed a long duration in circulation. The structures of the two major oligosaccharides from fetal bovine serum acetylcholinesterase and one major oligosaccharide from equine serum butyrylcholinesterase were determined. The three carbohydrate structures were of the biantennary complex type, but only the ones from fetal bovine serum acetylcholinesterase were fucosylated on the innermost N-acetylglucosamine residue of the core. Pharmacokinetic studies with native, desialylated, and deglycosylated forms of both enzymes indicate that the microheterogeneity in carbohydrate structure may be responsible, in part, for the multiphasic clearance of cholinesterases from the circulation of mice.

Expression of beta 1,6 branched asparagine-linked oligosaccharides in non-mitotic and non-migratory cells of normal human and rat tissues.

Malignant transformation of cells leads to the synthesis of large asparagine-linked oligosaccharides that exhibit a higher degree of beta 1,6 branching. In rodent and human tumor cell lines and certain human tumors, increased beta 1,6 branching of oligosaccharides has been shown to be associated with metastasis. In addition, this structural change occurs in glycoproteins of stimulated normal human lymphocytes. The leukoagglutinating Phaseolus vulgaris lectin (L-PHA) has a high affinity for tri- and tetraantennary beta 1,6 branches carrying oligosaccharides and has been widely used for the detection of such structures by histochemistry and blotting. We have analyzed a spectrum of normal human and rat tissues using a sensitive silver-intensified lectin-gold technique. Staining by L-PHA was detected in undifferentiated cells of germinative layers of the gastrointestinal and respiratory tract as well as testis. However, differentiated and non-mitotic epithelia in most organs showed strong lectin staining as well. Notable exceptions were the epithelium of the colon and resting mammary gland, which were unreactive with L-PHA. The histochemical studies were supplemented by lectin blotting, which showed the presence of diverse L-PHA-reactive glycoproteins in rat tissues. Our data may be of importance for the use of L-PHA in studies on human malignant tumors.

NMR evidence for a novel asparagine-linked oligosaccharide on cellobiohydrolase I from Trichoderma reesei RUTC 30

The primary structure of a novel phosphate-containing oligosaccharide, isolated from T. reesei cellobiohydrolase I, was determined by NMR techniques. The new compound has the same structure as GlcMan 7GlcNAc 2, but it is extended by one α-mannopyranosyl unit (Man-P) through a phosphate link. Three different heteronuclear ( 31P- 1H) NMR techniques were used to prove that the phosphate links the glycosidic site of Man-P with C-6 of unit Man-B. The presence of mannoses linked through a phosphate diester resembles glycosyl synthesis in yeast. © 1997 Federation of European Biochemical Societies.

Intracellular Disposal of Incompletely Folded Human α1-Antitrypsin Involves Release from Calnexin and Post-translational Trimming of Asparagine-linked Oligosaccharides

Protection of lung elastin fibers from proteolytic destruction is compromised by inefficient secretion of incompletely folded allelic variants of human alpha1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient sedimentation and coimmunoprecipitation techniques were employed to investigate quality control of human alpha1-antitrypsin secretion from stably transfected mouse hepatoma cells. The secretion-incompetent variant null(Hong Kong) (Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335) cannot fold into a functional conformation and was quantitatively associated with the molecular chaperone calnexin following biosynthesis. Assembly with calnexin required cotranslational trimming of glucose from asparagine-linked oligosaccharides. Intracellular disposal of pulse-radiolabeled molecules coincided with their release from calnexin. Released monomers and intracellular disposal were nonexistent in cells chased with cycloheximide, an inhibitor of protein synthesis. Post-translational trimming of asparagine-linked oligosaccharides and intracellular disposal were abrogated by 1-deoxymannojirimycin, an inhibitor of alpha-mannosidase activity, without affecting the monomer population. The data are consistent with a recently proposed quality control model (Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917) in which intracellular disposal requires dissociation from calnexin and post-translational trimming of mannose from asparagine-linked oligosaccharides.

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction.

The biosynthesis of complex asparagine (N)-linked oligosaccharides in vertebrates proceeds with the linkage of N-acetylglucosamine (GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:beta-D-mannoside beta 1-4 N-acetylglucosaminyltransferase III (GlcNAc-TIII, EC2.4.1.144) catalyzes the addition of GlcNAc to the mannose that is itself beta 1-4 linked to underlying N-acetylglucosamine. GlcNAc-TIII thereby produces what is known as a 'bisecting' GlcNAc linkage which is found on various hybrid and complex N-glycans. GlcNAc-TIII can also play a regulatory role in N-glycan biosynthesis as addition of the bisecting GlcNAc eliminates the potential for alpha-mannosidase-II, GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, and core alpha 1-6-fucosyltransferase to act subsequently. To investigate the physiologic relevance of GlcNAc-TIII function and bisected N-glycans, the mouse gene encoding GlcNAc-TIII (Mgat3) was cloned, characterized, and inactivated using Cre/loxP site-directed recombination. The Mgat3 gene is highly conserved in comparison to the rat and human homologs and is normally expressed at high levels in mammalian brain and kidney tissues. Using fluorescence in situ hybridization (FISH), the Mgat3 gene was regionally mapped to chromosome 15E11, near the Scn8a sodium channel gene at 15F1. Following homologous recombination in embryonic stem cells and Cre mediated gene deletion, Mgat3-deficient mice were produced that lacked GlcNAc-TIII activity and were deficient in E4-PHA visualized GlcNAc-bisected N-linked oligosaccharides. Nevertheless, GlcNAc-TIII deficient mice were found to be viable and reproduced normally. Moreover, such mice exhibited normal cellularity and morphology among organs including brain and kidney. No alterations were apparent in circulating leukocytes, erythrocytes or in serum metabolite levels that reflect kidney function. We thus find that GlcNAc-TIII and the bisecting GlcNAc in N-glycans appear dispensable for normal development, homeostasis and reproduction in the mouse.

Loss of glycosylation at Asn144 alters the substrate preference of the N8 influenza A virus neuraminidase.

Role of asparagine-linked (N-linked) oligosaccharide side chains in the maturation and the function of influenza virus neuraminidase (NA) subtype N8 was examined by site-directed mutagenesis and vaccinia virus expression system. Mutations in the consensus sequence for N-linked glycosylation at Asn 84 or 398 prevent the proper maturation of mutant NAs. On the contrary, mutation at Asn 144, that is conserved in all except two strains of influenza virus NA ever sequenced, did not affect the proper maturation and the transport of the mutant NA to the cell surface. Furthermore, this mutation led the alternation of substrate preference of this enzyme. These observations indicate that N-glycosylation at Asn 144 of N8 NA may be conserved from the functional requirement, but not from the structural necessity.

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin beta-subunit by disruption of disulfide bonds.

The human chorionic gonadotropin beta-subunit (hCGbeta) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGbeta produced in Chinese hamster ovary (CHO) cells: wild-type hCGbeta (betaWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of betaWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled betaWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGbeta molecule.

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis.

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.

Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.

Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.