Urinary Bladder Glycoproteins of the Rabbit: Extraction, Biochemical and Immunological Studies

Callahan, Hugh J.; Byrne, Dolores; Fritz, Robert; Mulholland, S. Grant

doi:10.3109/08820138509052445

Cited by 22 publications

(5 citation statements)

References 15 publications

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“…Variations in the expression of sialic acid are known in a number of pathological conditions of human urinary bladder (Videira et al, 2009). Besides, sialic acid mediates the interactions between pathogens and urothelial cells: in some cases, the high negative charge of sialylated chains prevents bacterial adhesion (Callahan et al, 1985), whereas in others sialic acid is involved in recognition and adhesion (Sakarya et al, 2003;Anderson et al, 2010). GlcNAc in O-linked glycans (Yamashita et al, 1985;Osawa and Tsuji, 1987;Spicer and Schulte, 1992).…”

Section: Discussionmentioning

confidence: 99%

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

Mastrodonato

Mentino

Lopedota

et al. 2016

Microsc. Res. Tech.

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Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic-Acid Schiff, Alcian Blue pH 2.5, High-Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA-I, WGA, SBA, BSI-B4, ConA, AAA, UEA-I, LTA, LFA, MAA-II, SNA) combined with chemical and enzymatic pre-treatments (β-elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O-linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin-binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co-distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.

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Section: Discussionmentioning

confidence: 99%

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

Mastrodonato

Mentino

Lopedota

et al. 2016

Microsc. Res. Tech.

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“…The monosaccharide compositions of RBM 1 and RBM 2 are shown in The glycoproteins in the urine have been analyzed extensively in order to evaluate the disease process affecting the urinary bladder, kidneys and urogenital tract [35]. Several low molecular weight glycoproteins such as Tamm-Horsfall glycoprotein (THGP)/Uromodulin, GP51, Fetuin-A, beta 2-glycoprotein-1, C-reactive protein, Amyloid beta A4 protein, Alpha-1-inhibitor 3, Vitamin D-binding protein, Kallikrein 3, uronic-acid rich protein (UAP) are found abundantly in mammalian urine [14,20,[35][36][37][38][39]. However, the results of our studies reveal that the high molecular weight RBM 1 and RBM 2 mucin fractions we have characterized are distinct from these glycoproteins including THGP as well as the MUC1 mucins of human and rabbit bladder that we have previously reported [13,18].…”

Section: Resultsmentioning

confidence: 99%

“…One proposed function for MUC1 is the protection of the epithelium from extreme pH and osmolarity [13,19]. Another bladder glycoprotein, GP51, resembling that reported to be decreased in the urine of interstitial cystitis patients compared to urine of healthy individuals, has been purified from rabbit bladder [20]. Previously, our laboratory has purified a major mucin glycoprotein with a molecular mass of about 245 kDa from radio labeled rabbit bladder explants and identified it as the rabbit homolog of human MUC1 [14].…”

Section: Introductionmentioning

confidence: 99%

Characterization of High Molecular Weight Mucins of Rabbit Bladder

Muthusamy¹,

Gowda²,

Bhavanandan³

2008

TOGLYJ

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Epithelial mucin glycoproteins of bladder act as an effective barrier against invasion by pathogenic microorganisms and injury by toxic substances in urine. Although these glycoconjugates play important roles in the pathophysiology of bladder disorders such as intestinal cystisis, cancer, and urinary tract infections, they have not been characterized in detail either in humans or in animals. Rabbits could be useful for developing models for studying bladder disorders. In this study, we purified and partially characterized two major high molecular weight rabbit bladder mucin glycoproteins, designated RBM 1 and RBM 2 , found in urine. Consistent with their mucin characteristics, amino acid compositions showed have high levels of serine, glutamic acid, proline, glycine and alanine, which together comprise 34% and 42% of the total amino acids in RBM 1 and RBM 2 , respectively. Carbohydrate compositional analysis indicated that RBM 1 and RBM 2 consist of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), N-acetylneuraminic acid (NeuAc) and fucose (Fuc) in the molar ratio of 1.0: 0.82: 0.12: 0.30: 0.02 and 1.0: 1.03: 0.46: 0.16: 0.05, respectively; mannose (Man) was not detected in either mucin. Both mucin fractions were strongly reactive to wheat germ agglutinin, but not to Ca2 antibody specific to a human tumor mucin antigen (asialylated carbohydrate linked to protein core), suggesting that most of the galactosyl residues of oligosaccharides are sialylated. Together, the data suggest that rabbit mucin glycoproteins characterized here are distinctively different from MUC1 mucin glycoprotein found in human urine.

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“…For specificity assays, GP1 was isolated from rabbit bladder mucosa as previously described [1]. Briefly, frozen NZW rabbit bladders were obtained from Cocalico Biologics (Reamstown, Pa.).…”

Section: Preparation Of Human Gp1mentioning

confidence: 99%

“…Hybridoma lines were grown in RPMI 1640 medium minus serum supplemented with Nutridoma (Boehringer Mannheim BiochemiThis assay has also been described previously [1]. In brief, microtiter plates (Immunlon 2, Fisher Scientific, Malveru, Pa.) were coated overnight with human or rabbit GP1, rabbit serum albumin (RSA), or THP (0.5 p-g/100 gl pert well) diluted in 0.01 M NaHCO 3 buffer (pH 9.6) with 0.02% NaN 3.…”

Section: Purification Of Mabs To Gp1mentioning

confidence: 99%

Urinary tract glycoprotein: distribution and antigenic specificity

et al. 1994

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It has previously been demonstrated that the urinary tract contains a unique glycoprotein (GP1) that appears to serve a function in the clearance of pathogenic bacteria. We prepared a panel of monoclonal antibodies (mAbs) to human GP1 and examined its antigenic specificity and distribution in the human urinary tract. This was also observed in relation to another glycoprotein associated with infection, Tamm-Horsfall protein (THP), as well as to URO-5, a protein used in identifying the lower urinary tract. In enzyme-linked immunoadsorbent assays (ELISA), all of the GP1 mAbs reacted with GP1 prepared from both human urine and rabbit bladder mucosa. No immunoreactivity was observed with either THP mAbs or URO-5 mAbs. Western-blot analyses using GP1 mAbs with human urine GP1 preparations detected a single band of approximately 50-52 kDa. Human urinary tract tissues were also examined by immunohistochemical techniques using the GP1 mAbs, commercial THP, and URO-5 mAbs. In paraffin-embedded bladder sections, only GP1 mAbs reacted with the urothelium. Selective staining of distal collecting tubules, renal pelvis, and ureters was demonstrated for GP1 mAbs. Proximal convoluted tubules, loops of Henle, and Bowman's capsule failed to stain for GP1. In the same tissues, THP mAbs reacted with the thick and thin segments of medullary loops of nephrons (Henle's loops). URO-5 mAbs stained fresh frozen sections of bladder urothelium, distal collecting tubules, and portions of Henle's loops; however, they failed to stain paraffin-embedded tissue sections. These GP1 mAbs provide a new tool for biochemical and histochemical investigations of the mucin layer in both healthy and diseased urinary tract tissue.

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Urinary Bladder Glycoproteins of the Rabbit: Extraction, Biochemical and Immunological Studies

Cited by 22 publications

References 15 publications

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

Characterization of High Molecular Weight Mucins of Rabbit Bladder

Urinary tract glycoprotein: distribution and antigenic specificity

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