Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site-specific glycosylation in yeast

To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 --> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106.     

In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion

Detailed structure-function information about human lipoprotein lipase (LPL) is unavailable because it is difficult to purify large amounts of the enzyme for study. To circumvent this problem, we constructed an in vitro LPL expression vector. Human LPL cDNA was cloned and inserted into the expression vector p91023(B). After transfection of COS M-6 cells with the human LPL cDNA construct, LPL enzyme activity was detected in cell extracts and culture medium. Purified human apolipoprotein C-II caused a 5-fold stimulation of the recombinant human LPL expressed in vitro. Using site-specific mutagenesis, Ala residues were substituted for Asn residues at two potential N-linked glycosylation sites (positions 43 and 359) and at a third unrelated Asn (position 257) in the LPL cDNA. RNA blot analysis demonstrated the presence of a single mRNA species in COS cells transfected with wild-type and mutant LPL expression vectors. Intracellular and secreted LPL activity was absent in the construct containing an Ala for Asn mutation at position 43, whereas the same substitutions at positions 257 and 359 did not appreciably affect activity. LPL activity was also absent in another construct containing a Gln for Asn mutation at position 43. Quantitation of LPL protein mass concomitant with measurement of enzyme activity showed that substitution of Ala or Gln for Asn at position 43 resulted in the production of an enzymatically inactive protein which accumulated intracellularly but was not secreted into the culture medium. Our report represents an initial documentation of the expression of cloned human LPL in vitro and of the importance of Asn-43 for both enzyme activity and secretion.     

Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation

The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as theO-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.     

Substitution of asparagine for serine-406 of the immunoglobulin mu heavy chain alters glycosylation at asparagine-402

Previous work suggested that the substitution of Asn for Ser at position 406 of the mu heavy chain of mouse IgM results in aberrant glycosylation at Asn402. In order to characterise the apparently abnormal glycosylation process more precisely, the mutant and wildtype mu chains were fragmented by cleavage with cyanogen bromide, and the resulting glycopeptides were analysed further. Measurements of lectin binding specificity as well as glycosidase sensitivity suggest that the oligosaccharide at Asn402 of wildtype mu is a hybrid type which does not contain terminal alpha(2-6) or alpha(2-3) linked sialic acid. By contrast, the corresponding oligosaccharide on Asn402 of mutant mu is complex and contains terminal sialic acid linked alpha(2-6) to galactose. The structural features for specifying the abnormal glycosylation are present in monomeric mutant IgM.     

Minimal structural and glycosylation requirements for ST6Gal I activity and trafficking

The influence of N-linked glycosylation on the activity and trafficking of membrane associated and soluble forms of the STtyr isoform of the ST6Gal I has been evaluated. We have demonstrated that the enzyme is glycosylated on Asn 146 and Asn 158 and that glycosylation is not required for the endoplasmic reticulum to Golgi transport of the membrane-associated form of the STtyr isoform. In addition, N-linked glycosylation may stabilize the protein but is not absolutely required for catalytic activity in vivo. In contrast, soluble forms of the protein consisting of amino acids 64-403, 89-403, and 97-403 are efficiently secreted and active in their fully glycosylated forms, but retained in the endoplasmic reticulum and inactive in their unglycosylated forms. These results suggest that membrane associated and soluble forms of the STtyr protein have different requirements for N-linked glycosylation. Elimination of the oligosaccharide attached to Asn 158 in the full length STtyr single and double glycosylation mutants generates proteins that are not cleaved and secreted but stably localized in the Golgi, like the STcys isoform of the ST6Gal I. This stable Golgi localization is correlated with the observation that these two mutants are active in in vivo assays but inactive in in vitro assays of membrane lysates. We predict that removal of N-linked oligosaccharides leads to an increased ability of the STtyr protein to self-associate or oligomerize which subsequently allows more stable retention in the Golgi and increased aggregation and inactivity when membranes are lysed in the in vitro activity assays.     

Site-specific glycosylation analysis of human apolipoprotein B100 using LC/ESI MS/MS

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.     

Plasma membrane delivery of the gastric H,K-ATPase: the role of beta-subunit glycosylation

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase beta-subunit (YFP-beta) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase alpha-subunit (CFP-alpha) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-alpha, wild-type YFP-beta, and YFP-beta mutants lacking one or two of the seven beta-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-beta resulted in the plasma membrane localization of the protein, whereas the expressed CFP-alpha was retained intracellularly. When coexpressed, both CFP-alpha and YFP-beta were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-beta prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the beta-subunit are essential for the plasma membrane delivery of the beta-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the beta-subunits from different species, is not critical for plasma delivery of the protein.     

Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin

N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn123-Thr125) introduced into a position close to the existing O-glycosylation site (Ser126) had an additional O-linked carbohydrate chain and not an additional N-linked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.     

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

The G82S polymorphism promotes glycosylation of the receptor for advanced glycation end products (RAGE) at asparagine 81: comparison of wild-type rage with the G82S polymorphic variant

Interaction between the receptor for advanced glycation end products (RAGE) and its ligands amplifies the proinflammatory response. N-Linked glycosylation of RAGE plays an important role in the regulation of ligand binding. Two potential sites for N-linked glycosylation, at Asn(25) and Asn(81), are implicated, one of which is potentially influenced by a naturally occurring polymorphism that substitutes Gly(82) with Ser. This G82S polymorphic RAGE variant displays increased ligand binding and downstream signaling. We hypothesized that the G82S polymorphism affects RAGE glycosylation and thereby affects ligand binding. WT or various mutant forms of RAGE protein, including N25Q, N81Q, N25Q/G82S, and N25Q/N81Q, were produced by transfecting HEK293 cells. The glycosylation patterns of expressed proteins were compared. Enzymatic deglycosylation showed that WT RAGE and the G82S polymorphic variant are glycosylated to the same extent. Our data also revealed N-linked glycosylation of N25Q and N81Q mutants, suggesting that both Asn(25) and Asn(81) can be utilized for N-linked glycosylation. Using mass spectrometry analysis, we found that Asn(81) may or may not be glycosylated in WT RAGE, whereas in G82S RAGE, Asn(81) is always glycosylated. Furthermore, RAGE binding to S100B ligand is affected by Asn(81) glycosylation, with consequences for NF-κB activation. Therefore, the G82S polymorphism promotes N-linked glycosylation of Asn(81), which has implications for the structure of the ligand binding region of RAGE and might explain the enhanced function associated with the G82S polymorphic RAGE variant.     

Nascent chicken ovalbumin contains the functional equivalent of a signal sequence

Highly purified mRNA for chicken ovalbumin has been translated in a cell-free protein synthesizing system from rabbit reticulocytes in the presence or absence of EDTA-stripped microsomal membranes from dog pancreas. Nascent--but not completed--ovalbumin was transferred across the microsomal membrane, as demonstrated by cotranslational core glycosylation of ovalbumin nascent chains, by resistance to posttranslational proteolysis of only the glycosylated ovalbumin chains, and by cosedimentation with the membrane of exclusively the glycosylated form. Furthermore, nascent chains of bovine prolactin were observed to compete with nascent ovalbumin for transfer across the microsomal membrane. However, no competition for membrane sites was observed between nascent chains of rabbit globin and either nascent ovalbumin or prolactin. We interpret these results to suggest that nascent ovalbumin contains the functional equivalent of a signal sequence for transfer across membranes, and that membrane components involved in the segregation of secretory proteins with cleaved signal sequences also function in the segregation of ovalbumin.     

Mutation of the glycosylated asparagine residue 286 in human CLN2 protein results in loss of enzymatic activity

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by the deficiency of the lysosomal tripeptidyl peptidase-I encoded by CLN2. We previously detected in two LINCL patients a homozygous missense mutation, p.Asn286Ser, that affects a potential N-glycosylation site. We introduced the p.Asn286Ser mutation into the wild-type CLN2 cDNA and performed transient expression analysis to determine the effect on the catalytic activity, intracellular targeting, and glycosylation of the CLN2 protein. Expression of mutant p.Asn286Ser CLN2 in HEK293 cells revealed that the mutant was enzymatically inactive. Western blot analysis demonstrated that at steady state the amounts of expressed p.Asn286Ser CLN2 were reduced compared with wild-type expressing cells. The rate of synthesis and the sorting of the newly synthesized p.Asn286Ser CLN2 in the Golgi was not affected compared with wild-type CLN2 protein. The electrophoretic mobility of the immunoprecipitated mutant p.Asn286Ser CLN2 was increased by approximately 2 kDa compared with the wild-type CLN2 protein, whereas deglycosylation led to the generation of polypeptides of the same apparent size. The data suggest that mutant p.Asn286Ser CLN2 lacks one oligosaccharide chain resulting in enzymatic inactivation.     

Partial glycosylation of Asn2181 in human factor V as a cause of molecular and functional heterogeneity. Modulation of glycosylation efficiency by mutagenesis of the consensus sequence for N-linked glycosylation.

Coagulation factor V (FV) circulates in two forms, FV1 and FV2, having slightly different molecular masses and phospholipid-binding properties. The aim was to determine whether this heterogeneity is due to the degree of glycosylation of Asn(2181). FVa1 and FVa2 were isolated and digested with endoglycosidase PNGase F. As judged by Western blotting, the FVa2 light chain contained two N-linked carbohydrates, whereas FVa1 contained three. Wild-type FV and three mutants, Asn(2181)Gln, Ser(2183)Thr, and Ser(2183)Ala, were expressed in COS1 cells, activated by thrombin, and analyzed by Western blotting. Wild-type FVa contained the 71 kDa-74 kDa doublet, whereas the Asn(2181)Gln and Ser(2183)Ala mutants contained only the 71 kDa light chain. In contrast, the Ser(2183)Thr mutant gave a 74 kDa light chain. This demonstrated that the third position in the Asn-X-Ser/Thr consensus affects glycosylation efficiency, Thr being associated with a higher degree of glycosylation than Ser. The Ser(2183)Thr mutant FVa was functionally indistinguishable from plasma-purified FVa1, whereas Asn(2181)Gln and Ser(2183)Ala mutants behaved like FVa2. Thus, the carbohydrate at Asn(2181) impaired the interaction between FVa and the phospholipid membrane, an interpretation consistent with a structural analysis of a three-dimensional model of the C2 domain and the position of a proposed phospholipid-binding site. In conclusion, we show that the FV1-FV2 heterogeneity is caused by differential glycosylation of Asn(2181) related to the presence of a Ser rather than a Thr at the third position in the consensus sequence of glycosylation.     

Role of glycosylation in structure and stability of Erythrina corallodendron lectin (EcorL): a molecular dynamics study

The effect of glycosylation on structure and stability of glycoproteins has been a topic of considerable interest. In this work, we have investigated the solution conformation of the oligosaccharide and its effect on the structure and stability of the glycoprotein by carrying out a series of long Molecular dynamics (MD) simulations on glycosylated Erythrina corallodendron lectin (EcorL) and nonglycosylated recombinant Erythrina corallodendron lectin (rEcorL). Our results indicate that, despite the similarity in overall three dimensional structures, glycosylated EcorL has lesser nonpolar solvent accessible surface area compared to nonglycosylated EcorL. This might explain the experimental observation of higher thermodynamic stability for glycosylated EcorL compared to nonglycosylated EcorL. Analysis of the simulation results indicates that, dynamic view of interactions between protein residues and oligosaccharide is entirely different from the static picture seen in the crystal structure. The oligosaccharide moiety had dynamically stable interactions with Lys 55 and Tyr 53, both of which are separated in sequence from the site of glycosylation, Asn 17. It is possible that glycosylation helps in forming long-range contacts between amino acids, which are separated in sequence and thus provides a folding nucleus. Thus our simulations not only reveal the conformations sampled by the oligosaccharide, but also provide novel insights into possible molecular mechanisms by which glycosylation can help in folding of the glycoprotein by formation of folding nucleus involving specific contacts with the oligosaccharide moiety.     

Identification of disulfide bonds and site-specific glycosylation in chicken alpha1-acid glycoprotein by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Recently, we reported the amino acid sequence of chicken alpha1-acid glycoprotein (chicken alpha1-AGP) [Biochem. Biophys. Res. Commun. 295 (2002) 587]. In this study, we located the disulfide bonds and site-specific glycosylation in chicken alpha1-AGP using tryptic digests of carbamidomethylated chicken alpha1-AGP, carbamidomethylated completely deglycosylated chicken alpha1-AGP (cd-alpha1-AGP), and nonreduced denatured cd-alpha1-AGP by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Based on the detection of peptides mlz 3037.4 (amino acid sequences 69-76 plus 161-183) and 3453.3 (amino acid sequences 69-80 plus 161-183), the two disulfide bonds of chicken alpha1-AGP were determined to be located at Cys 6-Cys 146 and Cys 73-Cys 163. The results also showed that Asn 16, 70, 77, and 87 were fully glycosylated and that Asn 62 was partially glycosylated.     

N-Linked oligosaccharides on the meprin A metalloprotease are important for secretion and enzymatic activity, but not for apical targeting

The alpha and beta subunits of meprins, mammalian zinc metalloendopeptidases, are extensively glycosylated; approximately 25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse meprin A. Recombinant meprin alpha and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Gln were all secreted and sorted exclusively into the apical medium of polarized Madin-Darby canine kidney cells, indicating that no specific N-linked oligosaccharide acts as a determinant for apical targeting of meprin alpha. Several of the mutant proteins had decreased enzymatic activity using a bradykinin analog as substrate, and deglycosylation of the wild-type protein resulted in loss of 75-100% activity. Some of the mutants were also more sensitive to heat inactivation. In studies with agents that inhibit glycosylation processes in vivo, tunicamycin markedly decreased secretion of meprin, whereas castanospermine and swainsonine had little effect on secretion, sorting, or enzymatic properties of meprin. When all the potential glycosylation sites on a truncated form of meprin alpha (alpha-(1-445)) were mutated, the protein was not secreted into the medium, but was retained within the cells even after 10 h. These results indicate that there is no one specific glycosylation site or type of oligosaccharide (high mannose- or complex-type) that determines apical sorting, but that core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin alpha.     

N-linked glycosylation of rabies virus glycoprotein. Individual sequons differ in their glycosylation efficiencies and influence on cell surface expression.

Many eukaryotic proteins are modified by N-linked glycosylation, a process in which oligosaccharides are added to asparagine residues in the sequon Asn-X-Ser/Thr. However, not all such sequons are glycosylated. For example, rabies virus glycoprotein (RGP) contains three sequons, only two of which appear to be glycosylated in virions. To examine further the signals in proteins which regulate N-linked core glycosylation, the glycosylation efficiencies of each of the three sequons in the antigenic domain of RGP were compared. For these studies, mutants were generated in which one or more sequons were deleted by site-directed mutagenesis. Core glycosylation of these mutants was studied using two independent systems: 1) in vitro translation in rabbit reticulocyte lysate supplemented with dog pancreatic microsomes, and 2) transfection into glycosylation-deficient Chinese hamster ovary cells. Parallel results were obtained with both systems, demonstrating that the sequon at Asn37 is inefficiently glycosylated, the sequons at Asn247 and Asn319 are efficiently glycosylated, and the glycosylation efficiency of each sequon is not influenced by glycosylation at other sequons in this protein. High levels of cell surface expression of RGP in Chinese hamster ovary cells are seen with any mutant containing an intact sequon at Asn247 or Asn319, whereas low levels of cell surface expression are seen when the sequon at Asn37 is present alone; deletion of all three sequons completely blocks RGP cell surface expression. Thus, although core glycosylation at Asn37 is inefficient, it is still sufficient to support a biological function, cell surface expression. Future studies using mutagenesis of this model protein and its expression in these two well defined systems will aim to begin to unravel the rules governing core glycosylation of glycoproteins.     

N-linked glycosylation of G. mellonella juvenile hormone binding protein - comparison of recombinant mutants expressed in P. pastoris cells with native protein

Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G. mellonella, this protein is glycosylated only at one (Asn(94)) of the two potential N-linked glycosylation sites (Asn(4) and Asn(94)). To investigate the function of glycosylation, each of the two potential glycosylation sites in the rJHBP molecule was examined by site-directed mutagenesis. MS analysis revealed that rJHBP overexpressed in the P. pastoris system may appear in a non-glycosylated as well as in a glycosylated form at both sites. We found that mutation at position Asn(94) reduces the level of protein secretion whereas mutation at the Asn(4) site has no effect on protein secretion. Purified rJHBP and its mutated forms (N4W and N94A) have the same JH binding activities similar to that of hJHBP. However, both mutants devoid of the carbohydrate chain are more susceptible to thermal inactivation. It is concluded that glycosylation of JHBP molecule is important for its thermal stability and secretion although it is not required for JH binding activity.     

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.     

Temporal relationship of translation and glycosylation of immunoglobulin heavy and light chains.

The initial glycosylation of MPC 11 gamma 2b heavy chains occurs quantitatively in vivo when the nascent heavy chains reach a size of approximately 38 000 daltons. Nonglycosylated, completed MPC 11 heavy chains cannot be glycosylated in these cells. Other classes of mouse heavy chains (i.e., mu, alpha, and gamma 1) also appear to be glycosylated as nascent chains; nonglycosylated, completed heavy chains cannot be glycosylated by the cell in any of these cases. In contrast, variant MPC 11 cells synthesizing a heavy chain with a carboxy-terminal deletion appear to glycosylate some heavy chains prior to chain completion and some heavy chains after chain completion and release from the polysomes. Similar to the variant MPC 11 cells, MOPC 46B cells (which synthesize a kappa light chain containing an oligosaccharide attached to an asparagine located 28 residues from the amino terminus) glycosylate the majority of light chains after prior to chain completion but also some light chains after chain completion and release from the polysomes. In addition, it appears that, although completed MOPC 46B light chains can be glycosylated if they are present in a monomeric form, they cannot be glycosylated if they are present in a covalent dimeric form.