Kyoko Hotta

Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa.

Sulfated glycoproteins were extracted and purified from porcine stomach mucous scraping. Four sulfated glycoprotein fractions were separated and subsequently purified. These compounds always accompanied the apparent peptic inhibitory activity and consisted of 15-18% (w/w) protein. The carbohydrate portions contained an equimolar ratio of galactose and hexosamine (mainly glucosamine), together with lesser amounts of fucose and sialic acid. The sulfate content of the above fractions was 2-9% (w/w) of the total sulfated glycoprotein. The mode of inhibition of the sulfated glycoproteins to peptic activity was investigated and suggested that there was binding of the sulfated glycoproteins to the substrate of pepsin, making the substrate resistant to peptic activity. The sulfated glycoproteins neither bound pepsin at pH 1.8 nor inhibited the hydrolysis of a synthetic dipeptide substrate of pepsin. Desulfation of the sulfated glycoproteins resulted in the loss of both the inhibitory activity and the precipitate formation. The precipitation curve for sulfated glycoprotein and porcine serum albumin showed that both bound in varying proportions and suggests that both components are multivalent in this precipitate formation.

Histochemical Reactivity of Normal, Metaplastic, and Neoplastic Tissues to α-Linked N-Acetylglucosamine Residue-specific Monoclonal Antibody HIK1083

Monoclonal antibody (MAb) HIK1083, which is obtained by immunizing mice with a preparation of rat gastric mucins, has been shown to bind specifically to α-linked N-acetylglucosamine (α-GlcNAc). We investigated the specificity of MAb HIK1083 by immunostaining normal human organs, mucinous metaplasia of human pancreas, adenocarcinomas of human stomach, pancreas, and colon, and normal rat organs. The specificity was investigated by making comparisons with (a) a stain that labels Class III concanavalin A (ConA)-reactive mucin (Class III mucin), i.e., paradoxical ConA (PCS), and (b) staining with horseradish peroxidase (HRP)-conjugated Griffonia simplicifolia agglutinin II (GSA-II). In normal human and rat organs and in mucinous metaplasia of human pancreas, immunostaining with MAb HIK1083 and PCS showed similar specificities for mucins in glandular mucous cells. In adenocarcinoma of stomach and pancreas, GSA-II showed the most widespread positivity, PCS showed the least, and MAb HIK1083 showed a reactivity between those two extremes. Colon adenocarcinomas were labeled only with GSA-II. These results demonstrate that MAb HIK1083 could be a useful screening tool for Class III mucin in normal, metaplastic, and carcinoma tissues, and that the α-GlcNAc residue is one of the specific sugar residues found in Class III mucin.

Jelly coat substances of sea urchin eggs. I. Sperm isoagglutination and sialopolysaccharide in the jelly.

Abstract Jelly coat substances of Anthocidaris crassispina and Pseudocentrotus depressus were fractionated on a hydroxylapatite column into several components. However, the immunological test of Outhterlony suggests that in the case of Anthocidaris jelly, such fractions may not be homogeneous. These fractions, rich in sialic acid, poor in protein, and with very little sulfate, belong to the sialopolysaccharides. Since Anthocidaris sialopolysaccharides show similar amino acid histograms, the peptide portion is considered to form the backbone structure of these components. Jelly sialopolysaccharides tightly agglutinate the homologous sperm, and, generally speaking, there is some relationship between the sialic acid content and the sperm-agglutinating activity of the sialopolysaccharides.

Reactivity of Glomerular and Serum lgA1 to Jacalin in IgA Nephropathy

To analyze O-linked oligosaccharides (O-glycans) in the hinge region of IgA1 in IgA nephropathy (IgAN), the reactivity of IgA1 to jacalin, which specifically binds to O-glycans, was investigated. Initially, renal biopsy specimens from 5 patients with IgAN and 3 patients with other renal diseases were investigated in an immunofluorescence study with jacalin, monoclonal antihuman IgA1 and IgA2 antibodies. All of the renal biopsy specimens of IgAN and none of other renal diseases were positively stained by both FITC-labeled jacalin and monoclonal anti-IgA1 antibody. The glomerular staining patterns of FITC-jacalin were similar to those of the monoclonal anti-IgA1 antibody. IgA2 was negative in all specimens. Based on the positive reactivity of deposited IgA1 to jacalin, the binding ability of serum IgA1 to jacalin was evaluated by inhibition assay using D-galactose in patients with IgAN (n = 58), other primary glomerulonephritides (PGN) (n = 41), and healthy controls (n = 52). The frequencies of the patients with serum IgA1 having a high affinity for jacalin were significantly greater in IgAN (19/58, 32.8%) compared with the healthy controls (2/52, 3.8%) and other PGN (4/41, 9.8%). These results suggested that the increased reactivity of O-glycan(s) in the IgA1 hinge region to jacalin is due to an unusual glycosylation of serum IgA1 in IgAN.

Aggregated human serum immunoglobulin A1 induced by neuraminidase treatment had a lower number of O-linked sugar chains on the hinge portion

A part of human serum immunoglobulin A1(IgA1) was aggregated by treatment with neuraminidase. Aggregated IgA1 was separated from non-aggregated IgA1 by gel permeation chromatography. The prepared asialo-hinge glycopeptide (asialo-HGP) from both IgA1 subfractions was treated with beta-galactosidase to determine the number of beta-linked sugar chains attached on the hinge region. Removal of the galactose residue from asialo-HGP resulted in the HPLC separation of three major peaks. MALDI-TOFMS analysis of the glycopeptides also indicated the presence of three HGP components with three, four and five N-acetylgalactosamine (GalNAc) residues, respectively. Comparison of their relative content among the glycopeptide components showed a higher content of the HGP component with a lower number of GalNAc residues on aggregated IgA1. Thus, asialo-HGP prepared from aggregated IgA1 induced by neuraminidase treatment had an incomplete core structure of O-linked oligosaccharides. Especially, the result suggested that the reduced number of the attached O-linked oligosaccharides on IgA1 take part in phenomena such as self-aggregation of asialo-IgA1.

Analysis of glycoform of O-glycan from human myeloma immunoglobulin A1 by gas-phase hydrazinolysis following pyridylamination of oligosaccharides

A comparative study was made on the glycoform of O-glycan from human myeloma immunoglobulin A1. By gas-phase hydrazinolysis, O-glycan was released from its hinge portion. The released oligosaccharide was pyridylaminated and separated by a two-dimensional analytical method of gel filtration and reverse-phase HPLC. Four major pyridylamino derivatives (P1-P4) were obtained. The neutral component (P4) among them was identified as Gal beta 1,3GalNAc-PA by cochromatography with an authentic standard pyridylamino sugar. The desialylation of the other components indicated the largest P1 and middle size P2 components possibly corresponded to a disialylated structure, NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAc-PA, and a monosialylated component, NeuAc alpha 2,3Gal beta 1,3GalNAc-PA, respectively. The structural assignment of P3 is still incomplete. Four similar components were also detected in bovine fetuin whose relative content (P1:P2: P3:P:4) was 16:43:19:22. The relative content (%) of P1-P4 (glycoform) in IgA1 from the healthy control was 10.1 +/- 3.3, 48.2 +/- 4.6, 7.0 +/- 2.6, and 34.7 +/- 4.5. The glycoform of O-glycan on IgA1 thus appears the same for any individual. Analysis of IgA1 myeloma protein indicated glycoforms distinct from those of the healthy controls. The relative content of these component could be classed as 2:8:0:90 (Type I, only one case designated as Kita), 5:24:3:68 (Type II, seven cases), and 9:41:5:45 (Type III, four cases). Thus, the results for IgA1 myeloma protein indicate that at least three glycoforms of O-glycan are possible for the IgA1 hinge structure. However, only one glycoform was found in the healthy controls.

Release of oligosaccharides possessing reducing-end N-acetylgalactosamine from mucus glycoprotein in Streptomyces sp. OH-11242 culture medium through action of endo-type glycosidase

A crude enzyme preparation from a culture medium of Streptomyces sp. OH-11242 contained endo-alpha-N-acetylgalactosaminidase activity. The activity could be induced by the addition of purified porcine gastric mucin to the culture medium. Oligosaccharides corresponding to approximately 2-14 glucose units were detected in the culture medium and also in an incubated reaction mixture of crude enzyme preparation and mucus glycoprotein. The resulting product with N-acetylgalactosamine at the reducing terminal implied the presence of a new type of endo-glycosidase liberating not only Gal beta 1-3GalNAc but also other larger oligosaccharides by hydrolysis of the O-glycosidic linkage between GalNAc and Ser (Thr).

Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

glucurono hydrolase EC 3.2.1.31) was studied by sequential lectin affinity chromatography, fl-glucuronidase glycopeptides were obtained by extensive pronase digestion followed by N-/14C/ acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, the relative distribution of glycan structure types is proposed. The presence of complex bi-antennary and oligomannose type glycans was indicated by Concanavaline A-Sepharose chromatography. The absence of O-glycans, triand tetraantennary type* glycans was demonstrated by analysis of Concanavaline A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin, Ricinus communis agglutinin. The presence of fucosylated glycans was revealed by reaction with Lotus tetragonolobus lectin and Ulex europaeus lectin. The presence of hybrid or poly (Nacetyl-lactosamine) type glycans was examinated by wheat germ agglutinin chromatography.

Extraction method for preparing pyridylamino sugar derivatives and application to porcine gastric mucus glycoprotein analysis

For the sensitive detection of free sugars and oligosaccharides, the use of their pyridylamino derivatives has now found general acceptance. To remove excess 2-aminopyridine from this derivative in a reaction mixture, gel filtration and ion-exchange chromatography were conducted. It was found in the present study that contaminated 2-aminopyridine could be selectively removed from the reaction mixture by adjusting the pH with saturated sodium bicarbonate at above 8.5 followed by extraction with benzene. By using this method, fewer purification steps and less time are required, with minimum loss of pyridylamino sugar derivatives.

Comparative study on mucus glycoproteins in rat stomach and duodenum

The density of mucus glycoprotein compared to that of the corpus, antrum and duodenum was; 1.52, 1.49 and 1.57 g/ml respectively. Carbohydrate composition of gastrointestinal mucus glycoprotein consisted of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic acid. Ratios of carbohydrate composition among corpus, antral and duodenal mucus glycoproteins differed. The average length of an oligosaccharide was found to be about 12-13, 14 and 10 sugars in the corpus, antrum and duodenum, respectively. In the corpus, the amino acid content was found to have the following quantitative order: Thr greater than Ser greater than Glx = Pro; in the antrum: Thr greater than Ser greater than Glx; and in the duodenum: Thr greater than Ser greater than Pro. Corpus, antral and duodenal mucus glycoproteins have the blood-group A antigen; antral mucus glycoprotein in particular exhibited strong blood-group A activity.