Qualitative characterization of oligosaccharide chains present on the rat zona pellucida glycoconjugates.

Zona pellucida (ZP), the extracellular glycocalyx surrounding the mammalian oocyte, is believed to mediate species-specific sperm-egg interaction. Despite numerous studies on characterization of ZP glycoconjugates in several species, little or no information is available on the number and chemical nature of the various components of the rat ZP. In this study we have attempted the biochemical characterization of the rat ZP using endo- and/or exo-glycohydrolases. Intact eggs from superovulated rats were radioiodinated by the chloramine-T method, and the labeled ZP components were resolved on SDS-PAGE under nonreducing conditions. These studies show that the rat ZP consists of three components with apparent molecular masses of 205 kDa (ZP1), 119 kDa (ZP2), and 115 kDa (ZP3). Unlike mouse ZP2 and ZP3, which resolve as distinct components on SDS-PAGE, rat ZP2 and ZP3 show substantial overlap in their molecular sizes and isoelectric points. Treatment of the rat ZP components with exo- (neuraminidase and alpha-L-fucosidase) and/or endo- (endoglycosidase H, endoglycosidase F, N-glycanase, and O-glycanase) glycohydrolases indicated the following: 1) Both rat ZP2 and ZP3 contain N-linked oligosaccharide (OS) units as indicated by their sensitivity to endoglycosidase F and N-glycanase. 2) Treatment with N-glycanase caused a reduction in size of the rat ZP2 and ZP3 components by nearly 50% and 60%, respectively, indicating that the two ZP components are highly glycosylated. 3) Rat ZP3 was sensitive to O-glycanase, suggesting that this ZP component contains O-linked OS unit(s). 4) No evidence was obtained for the presence of fucosyl or sialyl residue(s) on the O-linked OS unit(s) of rat ZP3.(ABSTRACT TRUNCATED AT 250 WORDS)     

O-glycosylation of the coronavirus M protein. Differential localization of sialyltransferases in N- and O-linked glycosylation.

It has previously been shown that the M (E1) glycoprotein of mouse hepatitis virus strain A59 (MHV-A59) contains only O-linked oligosaccharides and localizes to the Golgi region when expressed independently. A detailed pulse-chase analysis was made of the addition of O-linked sugars to the M protein; upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three different electrophoretic forms could be distinguished that corresponded to the sequential acquisition of N-acetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (SA). A fourth and fifth form could also be detected which we were unable to identify. Following Brefeldin A treatment, the M protein still acquired GalNAc, Gal, and SA, but the fourth and fifth forms were absent, suggesting that these modifications occur in the trans-Golgi network (TGN). In contrast, in the presence of BFA, the G protein of vesicular stomatitis virus (VSV), which contains N-linked oligosaccharides, acquired Gal and fucose but not SA. These results are consistent with earlier published data showing that Golgi compartments proximal to the TGN, but not the TGN itself, relocate to the endoplasmatic reticulum/intermediate compartment. More importantly, our data argue that, whereas addition of SA to N-linked sugars occurs in the TGN the acquisition of both SA on O-linked sugars and the addition of fucose to N-linked oligosaccharides must occur in Golgi compartments proximal to the TGN. The glycosylation of the M protein moreover indicates that it is transported to trans-Golgi and TGN. This was confirmed by electron microscopy immunocytochemistry, showing that the protein is targeted to cisternae on the trans side of the Golgi and co-localizes, at least in part, with TGN 38, a marker of the TGN, as well as with a lectin specific for sialic acid.     

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The involvement of O-linked oligosaccharide chains of the sea urchin egg receptor for sperm in fertilization

Recent investigations on the sea urchin egg receptor for spermhave led to its sequencing and the demonstration that it isa 350 kDa glycoprotein. In the current study, the N- and O-linkedoligosaccharide chains were cleaved from the protein fractionatedon concanavalin A-agarose. The putative O-linked oligosaccharidechains that did not bind to the lectin were further fractionatedby anion-exchange chromatography. Using a competition bioassaythat measured the ability of these oligosaccharide chains toinhibit fertilization, it was found that the N-linked chainswere devoid of inhibitory activity. Rather, the inhibitory activitywas localized to the O-linked chains, with the most highly charged,sulphated chains showing the highest inhibitory activity. Thebioactive oligosaccharides were labelled by reduction and assayedfor binding to sperm. The results of the binding assay, coupledwith the fertilization bioassay, indicate that the oligosaccharidesinhibit fertilization by binding to acrosome-reacted sperm.The bioactive oligosaccharide lacked species specificity infertilization bioassays, unlike the intact receptor and a recombinantaglyco protein containing only the extracellular domain of thereceptor. Since previous work showed that the recombinant proteininhibits fertilization species specifically and binds to acrosome-reactedsperm, a two-step model of sperm-egg interaction is proposed.The first step is postulated to be a low-affinity ionic interactionof the sulphated O-linked oligosaccharide chains of the receptorwith sperm that is not species specific. This is followed bya high affinity, species-specific interaction of the sperm withone or more binding sits on the polypeptide chain of the receptor. fertilization oligosaccharide receptor sea urchin egg sea urchin sperm     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Ultrastructural localization of bronchial inhibitor in human airways using protein A-gold technique

Gold immunolabelling of human airways using a polyclonal antibody specific for the bronchial inhibitor (brI) demonstrates that this protease inhibitor is present in the serous cells of the bronchial glands. Most of the largest secretory granules (89%) are uniformly stained for brI whereas the smallest granules (diameter less than 80 nm) appear frequently (68%) devoid of brI.     

Estimation of luminal mucin content in rats by measurement of O-linked oligosaccharide chains and direct ELISA

Changes in the small intestinal mucin contents in rats were evaluated by two methods, viz., a newly established ELISA and a method based on the measurement of O-linked oligosaccharide chains (OSC) as a mucin marker. Significant correlation was observed between the values of ELISA-derived mucins and OSC. The results confirm the usefulness of measurement of OSC as an alternative method for mucin determination.     

Identification and subcellular localization of paracellin-1 (claudin-16) in human salivary glands

Salivary calcium plays an important role in the pathogenesis of dental caries and the bio-mineralization of dental enamel and exposed dentin. The cellular and molecular basis of calcium secretion by the human salivary glands is, however, poorly understood. Recently a transcellular transport of calcium by the acinus cells has been proposed. In this paper we looked for evidence for paracellular calcium transport by investigating the presence and cellular localization of paracellin-1 (claudin-16) that has been implied in paracellular magnesium and calcium transport in the kidney. At the mRNA level, using RT-PCR with primers of appropriate sequence, paracellin-1 mRNA could be found in human Glandula parotis, Glandula submandibularis, Glandula labialis and Glandula sublingualis samples. In addition, a splice variant was detected in three out of 15 glands consisting of exons one and five of the paracellin gene. In immunohistochemical studies paracellin-1 colocalised in the salivary excretory ducts with the tight junction proteins ZO-1 and occludin suggesting a potential role in paracellular calcium and magnesium transport. In the acini no such colocalisation was observed; paracellin was instead detected at the basal poles of the cells, between cells of the same acinus as well as between cells of neighboring acini. At this location paracellin-1 might act as selectivity filter for the paracellular movement of ions and water during stimulated secretion. Thus, both in the ducts and in the acini a paracellular transport of calcium appears possible. Whether it occurs at all and the extent to which it contributes to the overall salivary calcium secretion remains, however, to be determined.     

Structure of the oligosaccharide chains in human alpha 1-protease inhibitor.

Two glycopeptides were obtained from alpha 1-protease inhibitor after extensive pronase digestion and chromatography on Bio-Gel P-10 and concanavalin A-Sepharose. these glycopeptides were characterized by compositional analysis and sequential exoglycosidase digestion followed at each step by methylation analysis. The partially methylated alditol acetates obtained were resolved by gas chromatography and identified by mass spectrometry. The proposes structures of the oligosaccharide moieties of the glycopeptides are given below. (formula: see text) The relative amounts of the two glycopeptides isolated from concanavalin A-Sepharose suggest that each protein molecule contains four carbohydrate chains; one large chain (A) and three small chains (B).     

Lectin cytochemical characterization of the N- and O-linked oligosaccharides in the human rectum

The oligosaccharides of the mucus glycoproteins of the human rectum are important for the lubricant and protective role suggested for the rectal mucus. Changes in oligosaccharide composition are observed in several colon diseases, and some of these changes could be used as diagnostic and prognostic indicators. Thus, a previous knowledge of the normal mucus glycoproteins is necessary. The aim of the present study is the characterization of the oligosaccharides of the goblet cells and enterocytes of the human rectum. For this, a battery of 15 lectins, in combination with chemical and enzymatic deglycosylation procedures, was used. Our results suggest the presence of N-acetylglucosamine (GlcNAc), Man, Glc, N-acetylneuraminic acid (Neu5Ac)(2–6)- and Neu5Ac(2–3)-linked, N-acetylgalactosamine (GalNAc) and Gal(1–3)GalNAc in the oligosaccharides of the goblet cells. Moreover, N-linked oligosaccharides specifically contained Gal(1–4)GlcNAc, while AAA-positive Fuc was only detected in O-linked oligosaccharides. Some of these carbohydrates were only visualized after removal of N- or O-linked oligosaccharides, suggesting a high level of approximation between the oligosaccharide chains, that render the carbohydrate inaccessible to the lectins. Differences in the labelling pattern between the goblet cells of the surface epithelium and the upper half of the crypts, and those of the lower half of the crypts suggests a maturation process for the goblet cells, which modifies the oligosaccharide composition of the secreted glycoproteins, as they ascend throughout the crypts. This maturation process includes the incorporation of new carbohydrates (GlcNAc), and the masking (Neu5Ac(2–3)-linked) or unmasking (Glc and GalNAc) of others.     

The subcellular localization of ubiquinone in human neutrophils.

Copper and zinc K-edge-extended X-ray-absorption fine structures were measured for the metal sites of freeze-dried bovine superoxide dismutase and the model compounds tetrakis(imidazole)cupric nitrate and tetrakis(imidazole)zinc perchlorate. Detailed simulation of the spectra indicates that the copper site of the enzyme is best fit by co-ordination of four imidazole groups with Cu-N(alpha) distances of 0.198 nm (1.98 A). The zinc site is best fit by three imidazole groups at 0.201 nm (2.01 A) and an oxygen (from aspartate) at 0.203 nm (2.03 A).     

Ultrastructural histochemical localization of acid phosphatase in salivary glands of Drosophila melanogaster.

The ultrastructural histochemical localization of acid phosphatase in salivary glands of third instar larvae of Drosophila melanogaster has been studied. Using Gomori's lead phosphate method for acid phosphatase detection, the optimal incubation time in the reaction medium was determined to be 30 min. When glands having wild-type acid phosphatase activity are incubated for this time, deposition of the final reaction product is observed in essentially every lysosome and artifactual staining is minimal.     

Structure determination of the major N- and O-linked carbohydrate chains of the beta subunit from equine chorionic gonadotropin

The carbohydrate moieties of equine chorionic gonadotropin alpha and beta subunits were released from the protein backbones by successive treatments with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the beta subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-, di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly alpha 1-6 fucosylated at the Asn-bound GlcNAc residue and having alpha 2-6 and alpha 2-3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the alpha subunit. The major part of the O-linked carbohydrate chains, occurring solely in the beta subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the beta subunit.     

Ultrastructural localization of caspase-14 in human epidermis.

Caspase-14 has been implicated in the formation of stratum corneum because of its specific expression and activation in terminally differentiating keratinocytes. However, its precise physiological role and its protein substrate are elusive. We studied the ultrastructural localization of caspase-14 in human epidermis to compare its distribution pattern with that of well-characterized differentiation markers. Immunogold cytochemistry confirmed that caspase-14 is nearly absent in basal and spinous layers. In the granular, layer nuclei and keratohyalin granules were labeled with increasing intensity towards the transitional layer. Particularly strong caspase-14 labeling was associated with areas known to be occupied by involucrin and loricrin, whereas F-granules, occupied by profilaggrin/filaggrin, were much less labeled. A high density of gold particles was also present at the forming cornified cell envelope, including desmosomes. In corneocytes, intense labeling was both cytoplasmic and associated with nuclear remnants and corneodesmosomes. These observations will allow focusing efforts of biochemical substrate screening on a subset of proteins localizing to distinct compartments of terminally differentiated keratinocytes.     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAF-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc2Ga14) or blood group A (Fuc2(Ga1NAc3) (Ga14) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the 3-linked G1cNAc at branch points, whereas the 6-linked GlcNAc residue ultimately forms short side chains with a Fuc2 (Ga1NAc3) Gal4 G1cNAc6 structure in individuals with A blood group determinant.The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. {ie75-1} This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of a2-linked fucose to the 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.     

The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.