Analysis of the glycosylation and phosphorylation of the alpha-subunit of the lysosomal enzyme, beta-hexosaminidase A, by site-directed mutagenesis.

The glycosylation and subsequent phosphorylation of mannose residues is a pivotal modification during the biosynthesis of lysosomal enzymes. We have identified the sites of N-linked glycosylation and oligosaccharide phosphorylation on the alpha-subunit of beta-hexosaminidase and have determined the influence of the oligosaccharides on the folding and transport of the enzyme. The potential glycosylation sequences, either singly or in combination, were eliminated through site-directed mutagenesis of the cDNA. By expression of the mutant cDNAs in COS-1 cells, each of the three glycosylation sites on the alpha-subunit was found to be modified by an oligosaccharide. One of the three oligosaccharides was the preferred site of phosphorylation. The absence of any individual oligosaccharide did not diminish the expression of the catalytic activity associated with the alpha-chain, implying proper folding and assembly of subunits. A profound effect was observed, however, when all three oligosaccharides were absent. The unglycosylated alpha-subunit, resulting from genetic alteration of all three glycosylation sites or synthesis of the wild-type protein in the presence of tunicamycin, was catalytically inactive. It was found to be improperly folded into an insoluble aggregate, linked through inappropriate disulfide bonds. The unglycosylated protein was trapped in the lumen of the endoplasmic reticulum and was found in a complex with the Ig heavy chain-binding protein, BiP. The properties of the nonglycosylated, misfolded alpha-subunit were similar to some mutant alpha-subunits in Tay-Sachs disease patients. The results indicate that the oligosaccharides are essential, although not in a site-specific manner, for proper folding and cellular transport of the alpha-subunit.     

Phosphorylation of Asn-linked oligosaccharides located at novel sites on the lysosomal enzyme cathepsin D.

We have examined the phosphorylation of Asn-linked oligosaccharides introduced at seven novel sites on human cathepsin D to determine whether the location of an oligosaccharide on a lysosomal enzyme affects its ability to serve as a substrate for UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase), the enzyme that catalyzes the initial step in the biosynthesis of mannose 6-phosphate residues. The glycosylation sites were introduced into the cathepsin D cDNA by site-directed mutagenesis and were selected to be widely distributed over the surface of the molecule. When the constructs were expressed in Xenopus oocytes, the oligosaccharides at each glycosylation site were phosphorylated at levels considerably above background (19-70% phosphorylation versus < 0.4% for the secretory protein glycopepsinogen). However, oligosaccharides located closer to the essential components of the phosphotransferase recognition domain (lysine 203 and amino acids 265-292) were phosphorylated better than oligosaccharides located further away. Similar results were obtained for oligosaccharides at homologous sites on a pepsinogen/cathepsin D chimera containing only lysine 203 and residues 265-319 of cathepsin D, although the absolute levels of phosphorylation were lower. These results demonstrate that there is considerable flexibility in the placement of glycosylation sites on cathepsin D in terms of the ability of the oligosaccharides to serve as substrates for phosphotransferase, although oligosaccharides located closer to the phosphotransferase recognition determinant are preferentially phosphorylated.     

Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency

Summary Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.Abbreviations PD Pseudo arylsulfatase-A Deficiency - ARA Arylsulfatase-A     

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.     

Heparan N-sulfatase: in vitro mutagenesis of potential N-glycosylation sites

Heparan N-sulfatase cDNA contains five potential N-glycosylation sites at Asn positions 41, 142, 151, 264, and 413. We used site-directed mutagenesis, substituting the codon of asparagine for glutamine, to eliminate selected glycosylation sites and then performed expression studies in COS-7 cells to determine the influence on the catalytic activity, lysosomal targeting, and glycosylation-phosphorylation of the enzyme. Elimination of site 5 did not affect significantly enzyme activity; elimination of sites 2 and 4 gave a partial reduction, while elimination of sites 1 and 3 resulted in drastic reduction of catalytic activity (25 and 14%, respectively, of normal values), indicating that glycosylation of asparagine 41 and asparagine 151 is essential for catalysis and/or enzyme stability. Wild type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for acquisition of enzyme activity and/or for enzyme stability. Metabolic labeling of each mutant cDNA, transiently transfected into COS cells, showed that enzyme from mutants N142Q, N264Q, and N413Q appeared to be properly folded, as judged by its ability to be proteolytically processed to a lower molecular weight form, while enzyme from mutants N41Q and N151Q did not reach lysosomes. These studies confirm that the five glycosylation sites of heparan N-sulfatase are all functional and show that Asn 41 and Asn 151 have a role in protein folding and/or stability.     

Role of N-linked oligosaccharide flexibility in mannose phosphorylation of lysosomal enzyme cathepsin L

Mannose phosphorylation of N-linked oligosaccharides by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a key step in the targeting of lysosomal enzymes in mammalian cells and tissues. The selectivity of this process is determined by lysine-based phosphorylation signals shared by lysosomal enzymes of diverse structure and function. By introducing new glycosylation sites at several locations on the surface of mouse procathepsin L and modeling oligosaccharide conformations for sites that are phosphorylated, it was shown that the inherent flexibility of N-linked oligosaccharides can account for the specificity of the transferase for oligosaccharides at different locations on the protein. By using this approach, the physical relationship between the lysine-based signal and the site of phosphorylation of mannose residues was determined. The analysis also revealed the existence of additional independent lysine-based phosphorylation signals on procathepsin L, which account for the low level of phosphorylation observed when the primary Lys-54/Lys-99 signal is ablated. Mutagenesis of residues that surround Lys-54 and Lys-99 and demonstration of mannose phosphorylation of a glycosylated derivative of green fluorescent protein provide strong evidence that the cathepsin L phosphorylation signal is a simple structure composed of as few as two well placed lysine residues.     

Synthesis of a human lysosomal enzyme, beta-hexosaminidase B, using the baculovirus expression system

Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by isolation of a recombinant insect virus (baculovirus) containing the cDNA for the beta chain within the viral polyhedron gene and infection of Sf9 cells with this construct. That portion of the enzyme secreted into the medium, 50%, was purified with concanavalin A Sepharose and subsequent affinity chromatography to yield beta-hexosaminidase B that is 75% pure. The product has an N-terminal amino acid sequence, specific activity, and size (M(r) 62,000) similar to that of the enzyme present in cultured human fibroblasts. However, endo H sensitivity studies revealed that the oligosaccharide structures present on recombinant beta-hexosaminidase B differ from those found on the enzyme synthesized in the human system. In addition, these structures lack the mannose 6-phosphate recognition marker that targets degradative hydrolases to lysosomes. Despite these differences, recombinant beta-hexosaminidase B does serve as a specific substrate for the mannose phosphorylating enzyme, N-acetylglucosaminyl phosphotransferase. Furthermore, the oligosaccharide moieties phosphorylated in vitro match those phosphorylated in vivo, pointing to the conformational integrity of the recombinant enzyme. Generous amounts of easily obtained, easily purified, and properly folded beta-hexosaminidase B will facilitate physical structural analysis of the enzyme.     

Susceptibility to endoglycosidase F and H at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits

We have studied the differential susceptibility to endoglycosidase F and H of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue was incubated with D-[2-3H]mannose for 6 h. [3H]Man-labeled TSH and free alpha-subunits were obtained from homogenates using specific antisera and were digested with endoglycosidase F and H in their native states or after heat-denaturation and reduction in the presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that effects of endoglycosidase at the individual glycosylation sites could be determined. There was very little preferential cleavage by endoglycosidase H and F among the glycosylation sites of TSH subunits. Endoglycosidase F treatment of native free alpha-subunits showed slight preferential cleavage at Asn 82 of alpha-subunits after a 4 h incubation, whereas endoglycosidase H cleaved oligosaccharides equally well at Asn 56 and Asn 82. The Asn 82 oligosaccharide of native TSH heterodimers was also slightly preferentially cleaved by endoglycosidase F, but endoglycosidase H cleaved oligosaccharides equally well at all TSH glycosylation sites. Heat denaturation, reduction and the presence of detergent did not alter this slight preferential cleavage by endoglycosidase F at Asn 82 of alpha-subunits, suggesting that the primary structures of the TSH subunits in part influenced the efficiency of enzyme action at specific sites. Thus, the susceptibility to endoglycosidase F differs very slightly at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these small differences could be due to properties of either the enzyme or substrates.     

Examination of an area in beta-hexosaminidase B homologous to that in cathepsin D linked to lysosomal targeting.

A lysine-rich area in the beta subunit of beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) homologous to residues 189-203 in Cathepsin D, previously proposed as being critical for efficient lysosomal targeting, was identified. In vitro mutagenesis of the Lys residues was followed by COS-1 cell expression of enzymatic activity. The intracellular mutant beta-hexosaminidase B activity had a T1/2 at 60 degrees C similar to that of the wild type enzyme, indicating that this region is likely on the surface of the folded enzyme, as is the targeting domain of Cathepsin D. However, in the case of beta-hexosaminidase B, mutation of the Lys residues did not affect lysosomal compartmentalization. These data suggest that the hunt for the common protein signal that results in proper intracellular transport of lysosomal enzymes will not be straightforward and that Lys residues may not be an absolute requirement of the signal.     

Human alpha-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; also known as alpha-galactosidase B) is the lysosomal exoglycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (approximately 53%), hybrid (approximately 12%), and high mannose-type (approximately 33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (approximately 53%). Approximately 80% of the complex oligo-saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with alpha-2,3-linked sialic acid residues. The majority of hybrid type oligo-saccharides were GalGlcNAcMan(6)GlcNAc-Fuc(0-1)GlcNAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man(5-7)GlcNAc(2) were the predominant species (approximately 90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of alpha-GalNAc and alpha-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that alpha-GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.     

The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor

We have analyzed the interaction of phosphorylated oligosaccharides and lysosomal enzymes with immobilized bovine liver cation-dependent mannose-6-P receptor. Oligosaccharides with phosphomonoesters were the only species that interacted with the receptor, and molecules with two phosphomonoesters showed the best binding. Lysosomal enzymes with several oligosaccharides containing only one phosphomonoester had a higher affinity for the receptor than did the isolated oligosaccharides, indicating the possible importance of multivalent interactions between weakly binding ligands and the receptor. The binding of a mixture of phosphorylated lysosomal enzymes to the cation-dependent Man-6-P receptor was markedly influenced by pH. At pH 6.3, almost all of the lysosomal enzymes bound to the receptor; whereas at pH 7.0-7.5, approximately one-third of the material passed through the column, one-third interacted weakly, and one-third bound tightly. The distribution of individual lysosomal enzyme activities was similar to that of the total material. The species of phosphorylated oligosaccharides present on the lysosomal enzymes which interacted poorly with the receptor were similar to those found on the tightly bound material and included species of oligosaccharides with two phosphomonoester groups. Isolated oligosaccharides of this type bound to the receptor over the entire pH range tested. These findings indicate that at neutral pH the phosphorylated oligosaccharides on some lysosomal enzyme molecules are oriented in a manner which makes them inaccessible to the binding site of the cation-dependent Man-6-P receptor. Since the same enzymes bind to the cation-independent Man-6-P receptor at neutral pH, at least a portion of the phosphomannosyl residues must be exposed. We conclude that small variations in the pH of the Golgi compartment where lysosomal enzymes bind to the receptors could potentially modulate the extent of binding to the two receptors.     

The effect of peptide deletions on the glycosylation of murine immunoglobulin M heavy chains

The glycosylation and processing of the asparagine-linked oligosaccharides at individual glycosylation sites on the mu-chain of murine immunoglobulin M were investigated using variant cell lines that synthesize and secrete IgM heavy chains with known peptide deletions. Normal murine IgM has five N-linked oligosaccharides in the constant region of each heavy or mu-chain. Each mu-chain has four complex-type oligosaccharides as well as a single high mannose-type oligosaccharide near the carboxyl terminus of the molecule. The peptide deletion of the C mu 1 constant region domain in the heavy chains synthesized by one variant cell line did not prevent subsequent glycosylation at more distal glycosylation sites. In fact, the presence of this deletion resulted in more complete glycosylation at the C-terminal glycosylation site. Evaluation of glycopeptides containing individual glycosylation sites by Concanavalin A-Sepharose indicated that this deletion had no significant effect on the processing of structures from high mannose-type to complex-type oligosaccharide chains. In contrast, a deletion of the C-terminal peptide region of the heavy chain of IgM synthesized by a second variant cell line resulted in intracellular processing to more highly branched oligosaccharide structures at several of the glycosylation sites not involved in the deletion.     

N-glycosylation is crucial for folding, trafficking, and stability of human tripeptidyl-peptidase I

Tripeptidyl-peptidase I (TPP I) is a lysosomal serine-carboxyl peptidase that sequentially removes tripeptides from polypeptides. Naturally occurring mutations in TPP I are associated with the classic late infantile neuronal ceroid lipofuscinosis. Human TPP I has five potential N-glycosylation sites at Asn residues 210, 222, 286, 313, and 443. To analyze the role of N-glycosylation in the function of the enzyme, we obliterated each N- glycosylation consensus sequence by substituting Gln for Asn, either individually or in combinations, and expressed mutated cDNAs in Chinese hamster ovary and human embryonic kidney 293 cells. Here, we demonstrate that human TPP I in vivo utilizes all five N-glycosylation sites. Elimination of one of these sites, at Asn-286, dramatically affected the folding of the enzyme. However, in contrast to other misfolded proteins that are retained in the endoplasmic reticulum, only a fraction of misfolded TPP I mutant expressed in Chinese hamster ovary cells, but not in human embryonic kidney 293 cells, was arrested in the ER, whereas its major portion was secreted. Secreted proenzyme formed non-native, interchain disulfide bridges and displayed only residual TPP I activity upon acidification. A small portion of TPP I missing Asn-286-linked glycan reached the lysosome and was processed to an active species; however, it showed low thermal and pH stability. N-Glycans at Asn-210, Asn-222, Asn-313, and Asn-443 contributed slightly to the specific activity of the enzyme and its resistance to alkaline pH-induced inactivation. Phospholabeling experiments revealed that N-glycans at Asn-210 and Asn-286 of TPP I preferentially accept a phosphomannose marker. Thus, a dual role of oligosaccharide at Asn-286 in folding and lysosomal targeting could contribute to the unusual, but cell type-dependent, fate of misfolded TPP I conformer and represent the molecular basis of the disease process in subjects with naturally occurring missense mutation at Asn-286.     

Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human beta-hexosaminidase

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.     

In vitro mutagenesis of potential N-glycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting.

The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways.     

Compartmental distribution of beta-hexosaminidase isoenzymes in I-cell fibroblasts.

A characteristic of the human lysosomal disorder I-cell disease is an abnormal excretion of most lysosomal hydrolases, including beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; beta-hexosaminidase) by cultured skin fibroblasts. Treatment of I-cell cultures with cycloheximide or tunicamycin demonstrated that (1) I-cell fibroblasts rapidly excrete all newly synthesized beta-hexosaminidase, (2) two qualitatively distinct pools of beta-hexosaminidase isoenzymes exist inside I-cell fibroblasts, one of which is a rapid-turnover excretory pool, and (3) the induction of an abnormal glycosylation of beta-hexosaminidase by tunicamycin in normal or I-cell fibroblast cultures does not affect subsequent excretion of the enzyme.     

Human acid beta-glucosidase: glycosylation is required for catalytic activity

The role of oligosaccharide modification in human acid beta-glucosidase function was investigated. This lysosomal enzyme has five putative N-glycosylation sites, four of which are occupied. The unglycosylated human protein was stable when expressed in bacteria or in Spodoptera frugiperda cells in the presence of tunicamycin but lacked catalytic activity. Deglycosylation of purified acid beta-glucosidase from human placenta with N-Glycanase under native conditions resulted in the removal of an accessible oligosaccharide chain from a single site with no effect on activity, whereas complete deglycosylation resulted in proportionate loss of activity. These studies demonstrate that occupancy of at least one glycosylation site is required for the formation and maintenance of acid beta-glucosidase in an active conformation.     

Influence of N-glycans on processing and biological activity of the nipah virus fusion protein

Nipah virus (NiV), a new member of the Paramyxoviridae, codes for a fusion (F) protein with five potential N-glycosylation sites. Because glycans are known to be important structural components affecting the conformation and function of viral glycoproteins, we analyzed the effect of the deletion of N-linked oligosaccharides on cell surface transport, proteolytic cleavage, and the biological activity of the NiV F protein. Each of the five potential glycosylation sites was removed either individually or in combination, revealing that four sites are actually utilized (g2 and g3 in the F(2) subunit and g4 and g5 in the F(1) subunit). While the removal of g2 and/or g3 had no or little effect on cleavage, surface transport, and fusion activity, the elimination of g4 or g5 reduced the surface expression by more than 80%. Similar to a mutant lacking all N-glycans, g4 deletion mutants in which the potential glycosylation site was destroyed by introducing a glycine residue were neither cleaved nor transported to the cell surface and consequently were not able to mediate cell-to-cell fusion. This finding indicates that in the absence of g4, the amino acid sequence around position 414 is important for folding and transport.     

Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking

The membrane glycoproteins Gn and Gc of Hantaan virus (HTNV) (family Bunyaviridae) are modified by N-linked glycosylation. The glycoproteins contain six potential sites for the attachment of N-linked oligosaccharides, five sites on Gn and one on Gc. The properties of the N-linked oligosaccharide chains were analyzed by treatment with endoglycosidase H, peptide:N-glycosidase F, tunicamycin, and deoxynojirimycin and were confirmed to be completely of the high-mannose type. Ten glycoprotein gene mutants were constructed by site-directed mutagenesis, including six single N glycosylation site mutants and four double-site mutants. We determined that four sites (N134, -235, -347, and -399) on Gn and the only site (N928) on Gc in their ectodomains are utilized, whereas the fifth site on Gn (N609), which faces the cytoplasm, is not glycosylated. The importance of individual N-oligosaccharide chains varied with respect to folding and intracellular transport. The oligosaccharide chain on residue N134 was found to be crucial for protein folding, whereas single mutations at the other glycosylation sites were better tolerated. Mutation at glycosylation sites N235 and N399 together resulted in Gn misfolding. The endoplasmic reticulum chaperones calnexin and calreticulin were found to be involved in HTNV glycoprotein folding. Our data demonstrate that N-linked glycosylation of HTNV glycoproteins plays important and differential roles in protein folding and intracellular trafficking.     

Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster

The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.