The epidermis of Timarete filigera (Polychaeta, Cirratulidae): histochemical and ultrastructural analysis of the gland cells

The aim of this study was to verify whether different living conditions of Polychaeta are correlated with morphological and functional differences in the organization of the integument. For this purpose, we decided to study the epidermis of Timarete filigera, a non-tubicolous polychaete. With this objective in mind, we have identified the various cellular types responsible for mucous secretion in the epidermis of this species and defined the histochemical composition of the mucus produced by different types of gland cells. Three types of gland cells have been identified by histochemical and ultrastructural studies in the epidermis of this polychaete. The histochemistry was carried out using standard techniques and peroxidase-labelled lectins. In type 1 cells, the secretory granules contain neutral glycoproteins with glucosidic residues of GalNAc, Galbeta 1,3 GalNAc, glucosidic and/or mannosidic residues. In type 2 cells, the secretory granules contain acid glycoproteins mainly sulphated with glucosidic residues of GalNAc, Galbeta 1,3 GalNAc, glucosidic and/or mannosidic residues, and some terminal sialic acid. In type 3 cells, the residual granules have the same chemical composition as that of granules present in type 2 cells. The secretion of these glandular mucous cells consists of mainly sulphated acidic glycoproteins and GAG resistant to testis jaluronidase. In these cells, the residual granules have the same chemical composition as that of their secretion. The heterogeneity of mucus composition may be correlated with its different functions.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Histochemical investigations on the secretory cells in the oesophagogastric tract of the Eurasian green toad,Bufo viridis

The secretory cells of the oesophagogastric tract of the Eurasian toad, Bufo viridis, were examined using standard histochemical methods and lectin histochemistry. Two goblet cell types were found in the oesophageal epithelium, differing in their morphology and the histochemical features of the secretory granules. These contained mainly acidic glycoconjugates, both sulphated and carboxylated, and a small amount of pepsinogen. Type I goblet cells contained stable class-III mucosubstances, which were absent in Type II. No pluricellular oesophageal glands were found. The oesophagogastric junction had a superficial epithelium similar to that of the oesophageal epithelium, with alveolar pluricellular glands, secreting stable class-III mucins, and few oxynticopeptic cells. The gastric mucosa presented secretory cells both in the surface epithelium and in the gastric glands. Superficial and foveolar cells produced neutral mucins with Gal1,3GalNAc residues. Neck cells, oxynticopeptic cells and endocrine cells were found in the gastric glands. Neck cells produced stable class-III mucosubstances. A functional gradient was observed in the oxynticopeptic cells from the oral to the aboral fundus, with a decrease in pepsinogen secretion towards the aboral fundus and a possible increase in HCl secretion. In the pyloric mucosa, the oxynticopeptic cells disappeared and the glands produced only neutral mucins, without stable class-III mucosubstances.     

Secretory cells in the clitellar epithelium of Eisenia foetida (Annelida,Oligochaeta): A histochemical and ultrastructural study

Eight secretory cell types are identified in the clitellar epithelium of Eisenia foetida, of which five have been described in detail previously (i.e., the large granular, fine granular, metachromatic, orthochromatic, and small granular proteinacecus cells). The remaining three secretory cell types are mucus-producing cells specific to the clitellar epithelium (type 3), cells associated with the chaetal follicles (type 4), and cells that occur exclusively in the tubercula pubertatis (type 5). Type 3 cells secrete a mucus containing neutral and acid mucosubstances. Ultrastructurally, type 3 cells are characterized by membrane-bound globules 0.4 to 3.7 μm in diameter. The contents of the globules have a finely reticulate appearance. The secretion of type 4 cells contains a collagenlike protein and neutral and sulfated acid mucosubstances. Type 4 cell secretory granules are membrane bound and range in diameter from 0.8 to 1.6 μm. They contain large, electron-dense, spheroid cores which are surrounded by parallel orientated microfibrils 14 nm in diameter. Type 5 cells give variable responses to the histochemical techniques used in the present study. An elastinlike protein is detected in about half of the type 5 cells and acid and neutral mucosubstances in the remainder. At the ultrastructural level the secretory granules vary in shape from spheroid to polygonal. Their finely, electron-dense contents exhibit progressive swelling which results in the eventual rupture of the limiting membranes of the granules. The necks of types 3, 4, and 5 cells contain a peripheral ring of microtubles (20 ± 1 nm in diameter).     

Distribution of sialoglycoconjugates in the oviductal isthmus of the horse during anoestrus, oestrus and pregnancy: a lectin histochemistry study

The distribution of sialic acid residues as well as other glycosidic sugars has been investigated in the horse oviductal isthmus during anoestrus, oestrus and pregnancy by means of lectin and pre-lectin methods. Ciliated cells and non-ciliated (secretory) cells exhibited different lectin binding profiles that were found to change during the investigated stages. Ciliated cells did not show any reactivity in the basal cytoplasm, while the supra-nuclear cytoplasm displayed a few of oligosaccharides with terminal and internal alphamannose (Man) and/or alphaglucose (Glc) during oestrus and pregnancy and a moderate presence of oligosaccharides terminating in alphafucose (Fuc) during oestrus; cilia exhibited a more complex glycoconjugate pattern for the presence of oligosaccharides terminating in N-acetylgalactosamine (GalNAc), GalNAcalpha1,3 GalNAcalpha1,3galactose(Gal)beta1,4Galbeta1,4N-acetylglucosamine(GlcNAc), Fuc, sialic acid (Neu5Ac)-aGalNAc belonging or not to the GalNAca1,3GalNAca1,3 Galb1,4 Galb1, 4GlcNAc sequence, and. alphaGalNAc and Neu5Aca 2,6Gal/GalNAc increased during oestrus. Cilia displayed terminal Galbeta1,3 GalNAc in pregnancy, terminal alphaGal in anoestrus and pregnancy and terminal or internal D-GlcNAc during anoestrus and pregnancy, respectively. The whole cytoplasm of non-ciliated cells showed oligosaccharides terminating with alphaGalNAc, Neu5Aca2,6Gal/GalNAc, Neu5Ac GalNAca 1,3GalNAcalpha1,3Galbeta1,4Galbeta1,4GlcNAc during the investigated stages, as well as GlcNAc in anoestrus and pregnancy. The supra-nuclear zone of non-ciliated cells exhibited oligosaccharides with terminal Galbeta1,4GlcNAc and internal Man during oestrus and pregnancy as well as terminal alphaGal and Fuc in oestrus and Neu5Ac-Galbeta1,3GalNAc in pregnancy. The luminal surface of non-ciliated cells showed glycans terminating with alphaGalNAc and/or Neu5Ac GalNAcalpha1,3 GalNAcalpha1,3Galbeta1,4Galbeta1,4GlcNAc in all specimens, oligosaccharides with terminal Galbeta1,4GlcNAc and internal Man during oestrus and pregnancy, Neu5Ac alpha2,6Gal/GalNAc in anoestrus and oestrus, and glycans terminating with Galbeta1,3GalNAc, Neu5A acalpha2,3 Galbeta1, 4GlcNac, Neu5ac-Galbeta1,3GalNAc, Neu5Ac-Galbeta1,4 GlcNAc in pregnancy. These findings show the presence of sialoglycoconjugates in the oviductal isthmus of the mare as well as the existence of great modifications in the glycoconjugates linked to different physiological conditions.     

A whole range of fine structural criteria for immunohistochemically identified LH cells in rats

Summary Pituitaries from normal, young and adult male rats were fixed either in sublimate-formalin or in glutaraldehyde-osmium. In adjacent Paraplast sections, almost all the gonadotrophs were immunostained with both LH and FSH antisera. The rat LH and FSH antisera used were shown to be highly specific by the absorption test and by double antibody radioimmunoassay. Thin and thick adjacent Epon sections were prepared for EM and immunohistochemical examination. Cells stained with the rat LH antiserum were identified by LM, and then observed in detail by EM. On the basis of these observations we suggest that the LH cells are arranged in a sequence of basophils, i.e., Types II/III, III, III/IV and IV: Type II/III basophils are elongate with a cytoplasmic process and less vesiculated. They have morphological features of Type II (classical thyrotrophs) and also of Type III basophils. Type III basophils are oval in shape and moderately vesiculated. Both Types II/III and III basophils can be divided into two classes of cell characterized mainly by the existence of only small secretory granules (150–220 nm in diameter) (Type A) or by the coexistence of small and large (350–500 nm) (Type B). Type III/IV basophils are cells intermediate between types III and IV basophils, and moderately vesiculated with an abundance of secretory granules (150–300 nm in diameter). Type IV basophils are large, spherical or oval cells whose RER cisternae are conspicuously dilated; they contain less numerous secretory granules (150–300 nm in diameter). It is concluded that LH cells are not a single cell type, but include a wide range of subtypes.     

Cytochemical localization of GalNAc and GalNAcβ1,4Galβ1,4 disaccharide in mouse zona pellucida

Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcş,4Galş,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcş,4Galş,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcş,4Galş,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcş,4Galş,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcş,4Galş,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.     

Chemical and Immunological Characterization of Galactosyl-β1-3-Globoside in Bovine, Human, and Rat Brain

Abstract: Our studies of bovine brain neutral glycosphingolipids (Ngsls) have revealed the presence of several short-chain (containing -CHO 1–4) and previously uncharacterized long-chain (−CHO > 4–5) Ngsls. We reported the structural characterization of brain GgOse₄Cer (GA1) and have now purified another brain Ngsl to homogeneity. The purified Ngsl migrated close to standard GgOse₄Cer and nLcOse₅Cer on a TLC plate employing two different solvent systems. The carbohydrate molar composition indicated the presence of Gal/Glc/GalNAc in a ratio of 2.8:1.0:0.9. Five permethylated alditol acetate peaks were characterized as 2,3,4,6-OMe₄Gal, 2,4,6-OMe₃Gal, 2,3,6-OMe₃Gal, 2,3,6-OMe₃Glc, and 4,6-OMe₂GalNAcMe by gas chromatography-mass spectrometry. The anomeric carbohydrate sequence has been determined by specific exoglycosidase digestion. Six-hundred megahertz ¹H NMR spectroscopy of the oligosaccharide released by ceramide glycanase hydrolysis confirmed the structure of the Ngsl as Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc\1-1Cer or IV³GalGbOse₄Cer. Using the immunooverlay technique with anti-stage-specific embryonic antigen 3 antibody, it was found in bovine, rat, and normal adult human brain and bovine myelin, but not in human or rat myelin.     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Loss of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 and reduced O-glycosylation in colon carcinoma cells selected for hepatic metastasis

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITC-PTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.     

Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.     

Histochemical study of glycoconjugates in the toadfish Halobatrachus didactylus oesophagus epithelium

The carbohydrate expression in the epithelium lining the oesophagus of the toadfish Halobatrachus didactylus was studied by means of conventional and lectin histochemistry. The stratified epithelium was constituted by basal cells, polymorphous cells in the intermediate layer, pyramidal and flattened cells in the outer layer and contained two types of large secretory cells: goblet cells and sacciform cells. PAS, Alcian blue pH 2.5 and pH 1.0 stained very strongly the goblet cells, weakly the surface of the other epithelial cells but did not stain the sacciform cells. The goblet cells cytoplasm contained oligosaccharides with terminal Galbeta1,3GalNAc, alpha/betaGalNAc, Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc residues (PNA, SBA, RCA120, UEA I, LTA and KOH-sialidase-WGA affinity). Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc were also found in the glycocalyx. The sacciform cells expressed sialyloligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acbeta2,6Gal/GalNAc, Neu5AcForssman pentasaccharide (MAL II, SNA, KOH-sialidase-DBA staining) as well as asialo-glycoconjugates with terminal/internal alphaMan (Con A affinity) and with terminal Galbeta1,3GalNAc, Forssman pentasaccharide, Galbeta1,4GlcNAc, GalNAc (HPA and SBA reactivity), alphaGal (GSA I-B4 reactivity), D-GlcNAc (GSA II labelling), alphaL-Fuc. The basal cells cytoplasm exhibited terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc. Intermediate cells showed oligosaccharides with terminal/internal alphaMan and/or terminating with Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc in the cytoplasm and with Neu5Acalpha2,3Galbeta1,4GlcNac, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc in the glycocalyx. The pyramidal cells expressed terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalbeta1,4NAc, alphaGal, alphaL-Fuc in the entire cytoplasm, terminal Neu5Acalpha2,3Galbeta1,4GlcNac and Forssman pentasaccharide in the apical extension, internal betaGlcNAc and/or terminal alphaL-Fuc in the luminal surface, Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alphaGal in the basolateral surface. The flattened cells displayed glycans with terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalNAc, alphaGal, D-GlcNAc in the entire cytoplasm, glycans terminating with Galbeta1,3GalNAc and/or internal betaGlcNAc in the sub-nuclear cytoplasm.     

The Arabidopsis thaliana putative sialyltransferase resides in the Golgi apparatus but lacks the ability to transfer sialic acid

A common feature of the animal sialyltransferases (STs) is the presence of four conserved motifs, namely large (L), small (S), very small (VS) and motif III. Although sialic acid (SA) has not been detected in plants, three orthologues containing sequences similar to the ST motifs have been identified in the Arabidopsis thaliana L. database. In this study, we report that the At3g48820 gene (Gene ID: 824043) codes for a Golgi resident protein lacking the ability to transfer SA to asialofetuin or Galβ1,3GalNAc and Galβ1,4GlcNAc oligosaccharide acceptors. Restoration of deteriorated motifs S, VS and motif III by constructing chimeric proteins consisting of the 28–308 amino acid region of the A. thaliana At3g48820 ST-like protein and the 264–393 amino acid region of the Oryza sativa L. AK107493 ST-like protein, or of the 28–240 amino acid region of the At3g48820 protein and the 204–350 amino acid region of the Homo sapiens L. α2,3-ST ( NP_008858 ) was not able to recover sialyltransferase activity. Altering the appropriate amino acid regions of the A. thaliana At3g48820 ST-like protein to those typical for the mammalian motif III (HHYWE) and VS motif (HDADFE) also did not have any effect. Our data, together with previous results, indicate that A. thaliana in particular, and plants in general, do not have transferases for SA. Substrates for the plant ST-like proteins might be compounds involved in secondary metabolism.     

O-glycosylation potential of lepidopteran insect cell lines

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.     

Determination of the site-specific oligosaccharide distribution of the O-glycans attached to the porcine submaxillary mucin tandem repeat. Further evidence for the modulation of O-glycans side chain structures by peptide sequence.

Little is known of the degree that polypeptide sequence and the local environment modulate the structures of O-linked glycans. Toward this understanding, the site-specific mono- (GalNAc-O-), di- (beta-Gal-1,3-alpha-GalNAc-O-), and trisaccharide (alpha-Fuc-1,2-beta-Gal-1,3-alpha-GalNAc-O-) distributions have been determined for 29 of the 31 O-glycosylated Ser/Thr residues in the tandem repeat domains of blood group A-negative porcine submaxillary gland mucin. The glycosylation patterns obtained from three individual animals are in agreement with earlier incomplete determinations on a pooled mucin (Gerken, T. A., Owens, C. L., and Pasumarthy, M. (1997) J. Biol. Chem. 272, 9709-9719; Gerken, T. A., Owens, C. L., and Pasumarthy, M. (1998) J. Biol. Chem. 273, 26580-26588), confirming that the addition of the peptide-linked GalNAc and its substitution by beta-1,3-Gal are sensitive to local peptide sequence in a highly reproducible manner in vivo. The present data further support earlier suggestions of an inverse correlation of the density of hydroxyamino acid residues (and by inference the density of peptide GalNAc) with the extent of substitution of the peptide-linked GalNAc by beta-1,3-Gal. This effect is highly correlated for Ser-linked glycans but not for Thr-linked glycans. A similar correlation is observed with respect to the in vivo peptide GalNAc glycosylation pattern. In contrast, the addition of alpha-1,2-Fuc to beta-Gal shows no apparent correlation with hydroxyamino acid density, although a marked elevation in the fucosylation of Ser-linked glycans compared with Thr-linked glycans is observed. The above effects may represent both steric and conformational factors acting to alter the relative accessibility and activity of the glycosyltransferases toward substrate. These results demonstrate that the porcine submaxillary gland core 1 beta 3-galactosyltransferase and alpha2-fucosyltransferase exhibit unique peptide/glycopeptide sensitivities that may provide mechanisms for the modulation of O-linked side chain structures.     

Alteration of cell surface carbohydrates associated with ordered and disordered proliferation of oral epithelia: a lectin histochemical study in oral leukoplakias, papillomas and carcinomas

Abstract. Cell surface carbohydrates in healthy oral mucosa (n = 15), leukoplakias without ( n = 48) and with (n = 62) dysplasia, oral papillomas (n = 6) and squamous cell carcinomas (SCCs) (n – 40) were examined using the lectins peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEA I), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), and Griffonia simplicifolia agglutinin I (GS I-B₄). Binding of these lectins in formalin-fixed, paraffin-embedded tissues was demonstrated using either the peroxidase-anti-peroxidase (PAP) method or the avidin-biotin method. Healthy oral epithelia revealed binding sites for these lectins mostly in the suprabasal keratinocytes with occasional PNA binding also in their basal cells. Unlike healthy mucosa, a number of leukoplakias without and with dysplasia revealed receptor sites for UEA I also in their basal layer. Only those keratinocytes undergoing squamoidal differentiation exhibited SBA binding. Staining patterns of UEA I and SBA did not vary significantly between either leukoplakias without and with dysplasia or papillomas and SCCs. Conversely, a reduction or lack of binding sites for PNA (Galβl-3GalNAc), HPA (D-GalNAcα) and GS I-B₄ (αD-Gal) was observed more frequently in leukoplakias with dysplasia and SCCs contrasting their counterparts lacking epithelial dysplasia.
Cell surface glycosyl residues play an important role in the regulation of cell proliferation and epithelial growth. Aberrant glycosylation in oral dysplastic leukoplakias and carcinomas leading to the lack of the relevant terminal sugar residues from their cell surface carbohydrates is probably a major reason for the hyper-/ disordered proliferation.     

Syntheses of mucin-type <Emphasis Type="Italic">O</Emphasis>-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of <Emphasis Type="Italic">Bifidobacterium</Emphasis> endo-α-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminidase

Endo-α-N-acetylgalactosaminidase catalyzes the release of Galβ1-3GalNAc from the core 1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr) of mucin glycoproteins and synthetic p-nitrophenyl (pNP) α-linked substrates. Here, we report the enzymatic syntheses of core 1 disaccharide-containing glycopeptides using the transglycosylation activity of endo-α-N-acetylgalactosaminidase (EngBF) from Bifidobacterium longum. The enzyme directly transferred Galβ1-3GalNAc to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T and MUC1a when Galβ1-3GalNAcα1-pNP was used as a donor substrate. The enzyme was also found to catalyze the reverse-hydrolysis reaction. EngBF synthesized the core 1 disaccharide-containing oligosaccharides when the enzyme was incubated with either glucose or lactose and Galβ1-3GalNAc prepared from porcine gastric mucin using bifidobacterial cells expressing endo-α-N-acetylgalactosaminidase. Synthesized oligosaccharides are promising prebiotics for bifidobacteria.     

Lectin histochemistry of epidermal glandular cells in the earthworm Lumbricus terrestris (Annelida Oligochaeta)

Carbohydrate residues were localized in the glandular cells of the epidermis of Lumbricus terrestris by lectin histochemistry. The following biotinylated lectins were used: ConA, PNA, WGA, UEA-I. Each lectin has a specific binding pattern in the epidermal glandular cells. The ConA binding is evident in the orthochromatic mucous cells; PNA in the metachromatic mucous cells; WGA in the neuroendocrine-like cells; UEA-I in the cuticle. The epidermal glandular cells possess specific sites for the different lectins in relation to their functional characteristics. Therefore, these sugar residues indicate different behaviours of the cells in epidermal functions related to ion transport, receptor-secretory processes and defence.