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• W1970480898 abstract "Tamm-Horsfall glycoprotein (THP), the most abundant protein in mammalian urine, has been implicated in defending the urinary tract against infections by type 1-fimbriated Escherichia coli. Recent experimental evidence indicates that the defensive capability of THP relies on its single high mannose chain, which binds to E. coli FimH lectin and competes with mannosylated uroplakin receptors on the bladder surface. Here we describe several major differences, on both structural and functional levels, between human THP (hTHP) and pig THP (pTHP). pTHP contains a much higher proportion (47%) of Man5GlcNAc2 than does hTHP (8%). FimH-expressing E. coli adhere to monomeric pTHP at an approximately 3-fold higher level than to monomeric hTHP. This suggests that the shorter high mannose chain (Man5GlcNAc2) is a much better binder for FimH than the longer chains (Man6–7GlcNAc2) and that pTHP is a more potent urinary defense factor than hTHP. In addition, unlike hTHP whose polyantennary glycans are exclusively capped by sialic acid and sulfate groups, those of pTHP are also terminated by Galα1,3Gal epitope. This is consistent with the fact that the outer medulla of pig kidney expresses the α1,3-galactosyltransferase, which is completely absent in human kidney. Finally, pTHP is more resistant to leukocyte elastase hydrolysis than hTHP, thus explaining why pTHP is much less prone to urinary degradation than hTHP. These results demonstrate for the first time that the species variations of the glycomoiety of THP can lead to the differential binding of THP to type 1-fimbriated E. coli and that the differences in high mannose processing may reflect species-specific adaptation of urinary defenses against E. coli infections. Tamm-Horsfall glycoprotein (THP), the most abundant protein in mammalian urine, has been implicated in defending the urinary tract against infections by type 1-fimbriated Escherichia coli. Recent experimental evidence indicates that the defensive capability of THP relies on its single high mannose chain, which binds to E. coli FimH lectin and competes with mannosylated uroplakin receptors on the bladder surface. Here we describe several major differences, on both structural and functional levels, between human THP (hTHP) and pig THP (pTHP). pTHP contains a much higher proportion (47%) of Man5GlcNAc2 than does hTHP (8%). FimH-expressing E. coli adhere to monomeric pTHP at an approximately 3-fold higher level than to monomeric hTHP. This suggests that the shorter high mannose chain (Man5GlcNAc2) is a much better binder for FimH than the longer chains (Man6–7GlcNAc2) and that pTHP is a more potent urinary defense factor than hTHP. In addition, unlike hTHP whose polyantennary glycans are exclusively capped by sialic acid and sulfate groups, those of pTHP are also terminated by Galα1,3Gal epitope. This is consistent with the fact that the outer medulla of pig kidney expresses the α1,3-galactosyltransferase, which is completely absent in human kidney. Finally, pTHP is more resistant to leukocyte elastase hydrolysis than hTHP, thus explaining why pTHP is much less prone to urinary degradation than hTHP. These results demonstrate for the first time that the species variations of the glycomoiety of THP can lead to the differential binding of THP to type 1-fimbriated E. coli and that the differences in high mannose processing may reflect species-specific adaptation of urinary defenses against E. coli infections. Escherichia coli is the major causative agent of urinary tract infection, the most common nonepidemic bacterial infection in humans and domestic animals (1.Mulvey M.A. Cell. Microbiol. 2002; 4: 257-271Crossref PubMed Scopus (272) Google Scholar). This pathogen enters the urinary tract by an ascending route from the intestinal flora, and the critical first step in colonization relies on bacterial binding to carbohydrate sequences carried by glycoproteins and glycolipids exposed at the luminal surface of the urinary tract (2.Beachey E.H. J. Infect. Dis. 1981; 143: 325-345Crossref PubMed Scopus (952) Google Scholar). This binding is mediated by lectin-like adhesins on the tip of E. coli fimbriae, which are classified according to their sugar specificity. Thus, type 1, P and S fimbriae recognize high mannose glycans, Galα1,4Galβ terminal disaccharide of glycolipids, and NeuAcα2,3Gal sequence-capping sialylated glycans, respectively (3.Sharon N. FEBS Lett. 1987; 217: 145-157Crossref PubMed Scopus (297) Google Scholar). Within the type 1 fimbriae, phenotypic variants of FimH adhesin have been identified, based on their binding affinity to high mannose glycans (4.Sokurenko E.V. Courtney H.S. Maslow J. Siitonen A. Hasty D.L. J. Bacteriol. 1995; 177: 3680-3686Crossref PubMed Google Scholar). Interestingly, FimH variants exhibiting low affinity binding to high mannose glycans (M1L) predominate in E. coli isolates from the large intestine, whereas those exhibiting high affinity to high mannose glycans (M1H) predominant in E. coli isolates from the urinary tract (5.Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (300) Google Scholar). These results suggest that there is a selective advantage in the urinary tract for the E. coli strains expressing a particular type of FimH adhesin.During the advanced stage of cellular differentiation, mammalian urothelium elaborates a group of integral membrane proteins called uroplakins (6.Wu X.R. Manabe M. Yu J. Sun T.T. J. Biol. Chem. 1990; 265: 19170-19179Abstract Full Text PDF PubMed Google Scholar, 7.Wu X.R. Sun T.T. J. Cell Sci. 1993; 106: 31-43Crossref PubMed Google Scholar, 8.Wu X.R. Lin J.H. Walz T. Haner M. Yu J. Aebi U. Sun T.T. J. Biol. Chem. 1994; 269: 13716-13724Abstract Full Text PDF PubMed Google Scholar, 9.Wu X.R. Sun T.T. Medina J.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9630-9635Crossref PubMed Scopus (261) Google Scholar). Together, these glycoproteins constitute the major protein building blocks of the asymmetric unit membrane, a rigid-looking structure that covers over 90% of the luminal surfaces of the proximal urethra, bladder, ureter, and renal pelvis (10.Koss L.G. Lab. Invest. 1969; 21: 154-168PubMed Google Scholar, 11.Walz T. Häner M. Wu X.R. Henn C. Engel A. Sun T.T. Aebi U. J. Mol. Biol. 1995; 248: 887-900Crossref PubMed Scopus (92) Google Scholar). Although uroplakin III carries complex-type N-glycans whose NeuAcα2,3Gal sequences may serve as receptors for S-fimbriated E. coli, uroplakins Ia and Ib carry a single high mannose glycan (7.Wu X.R. Sun T.T. J. Cell Sci. 1993; 106: 31-43Crossref PubMed Google Scholar, 12.Malagolini N. Cavallone D. Wu X.R. Serafini-Cessi F. Biochim. Biophys. Acta. 2000; 1475: 231-237Crossref PubMed Scopus (23) Google Scholar). In vitro experiments have demonstrated that type 1-fimbriated E. coli binds to uroplakins Ia and Ib in a mannose-specific fashion (9.Wu X.R. Sun T.T. Medina J.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9630-9635Crossref PubMed Scopus (261) Google Scholar). Infection of mouse bladders with type 1-fimbriated E. coli showed that the FimH-containing tip regions of type 1 fimbriae adhere to the central depression of asymmetric unit membrane (AUM) plaques, where uroplakin Ia and Ib reside (13.Mulvey M.A. Lopez-Boado Y.S. Wilson C.L. Roth R. Parks W.C. Heuser J. Hultgren S.J. Science. 1998; 282: 1494-1497Crossref PubMed Scopus (767) Google Scholar). These findings indicate that uroplakins serve as the major urothelial receptors for type 1-fimbriated E. coli.If the expression of adhesins recognizing various carbohydrates comes about as a natural selection for E. coli to adapt to specific habitats, the host itself, under selective pressure, has developed defense mechanisms against bacterial adhesion and colonization. In the respiratory and intestinal epithelium, for instance, the abundant mucus covering the epithelium prevents the adhesion of pathogens to glycoproteins and glycolipids exposed at the luminal membrane. This type of mucus defense is largely absent in the urothelial surface; however, it has been recently suggested that urinary defense against bacterial adhesion may depend on soluble glycoprotein receptors in the urine (1.Mulvey M.A. Cell. Microbiol. 2002; 4: 257-271Crossref PubMed Scopus (272) Google Scholar, 14.Kachar B. Liang F. Lins U. Ding M. Wu X.R. Stoffler D. Aebi U. Sun T.T. J. Mol. Biol. 1999; 285: 595-608Crossref PubMed Scopus (112) Google Scholar, 15.Pak J. Pu Y. Zhang Z.T. Hasty D.L. Wu X.R. J. Biol. Chem. 2001; 276: 9924-9930Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). In fact, the kidney cells of the thick ascending Henle's limb release into the urine from the GPI-anchored counterpart a protein at a rate of approximately 50 mg daily in humans, which is particularly rich in carbohydrates (30% of total weight) (16.Cavallone D. Malagolini N. Serafini-Cessi F. Biochem. Biophys. Res. Commun. 2001; 280: 110-114Crossref PubMed Scopus (73) Google Scholar, 17.Grant A.M. Neuberger A. Clin. Sci. 1973; 44: 163-179Crossref PubMed Scopus (33) Google Scholar, 18.Fletcher A.P. Neuberger A. Ratcliffe W.A. Biochem. J. 1970; 120: 417-424Crossref PubMed Scopus (105) Google Scholar, 19.Serafini-Cessi F. Malagolini N. Cavallone D. Am. J. Kidney Dis. 2003; 42: 658-676Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). This glycoprotein was first purified by Igor Tamm and Frank Horsfall (20.Tamm I. Horsfall F.L. Proc. Soc. Exp. Biol. Med. 1950; 74: 108-114Crossref Scopus (210) Google Scholar) from healthy individuals, hence the name Tamm-Horsfall glycoprotein (THP). 1The abbreviations used are: THP, Tamm-Horsfall glycoprotein; hTHP, human THP; pTHP, pig THP; ConA, concanavalin A; GS-IB4, G. simplicifolia isolectin B4; PNGase F, glycopeptidase F; PBS, phosphate-buffered saline; DEF, diatomaceous earth filter; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin. 1The abbreviations used are: THP, Tamm-Horsfall glycoprotein; hTHP, human THP; pTHP, pig THP; ConA, concanavalin A; GS-IB4, G. simplicifolia isolectin B4; PNGase F, glycopeptidase F; PBS, phosphate-buffered saline; DEF, diatomaceous earth filter; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin. It was later detected in the urine of all mammals studied, and the amino acid sequences predicted by cDNA show a high degree of homology among THPs from various species (21.Pennica D. Kohr W.J. Kuang W.J. Glaister D. Aggarwal B.B. Chen E.Y. Goeddel D.V. Science. 1987; 236: 83-88Crossref PubMed Scopus (214) Google Scholar, 22.Fukuoka S. Freedman S.D. Yu H. Sukhatme V.P. Scheele G.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1189-1193Crossref PubMed Scopus (89) Google Scholar, 23.Prasadan K. Bates J. Badgett A. Dell M. Sukhatme V. Yu H. Kumar S. Biochim. Biophys. Acta. 1995; 1260: 328-332Crossref PubMed Scopus (17) Google Scholar, 24.Yu H. Papa F. Sukhatme V.P. Gene Expr. 1994; 4: 63-75PubMed Google Scholar). The glycomoiety of human THP (hTHP) consists mainly of polyantennary N-glycans, but a single N-glycosylation bears high mannose sequences in both native THP and recombinant THP expressed in transfected cells (25.Hard K. Van Zadelhoff G. Moonen P. Kamerling J.P. Vliegenthart F.G. Eur. J. Biochem. 1992; 209: 895-915Crossref PubMed Scopus (273) Google Scholar, 26.Serafini-Cessi F. Dall'Olio F. Biochem. J. 1983; 215: 483-489Crossref PubMed Scopus (54) Google Scholar, 27.Serafini-Cessi F. Dall'Olio F. Malagolini N. Biosci. Rep. 1984; 4: 269-274Crossref PubMed Scopus (47) Google Scholar, 28.Serafini-Cessi F. Malagolini N. Hoops T.C. Rindler M.J. Biochem. Biophys. Res. Commun. 1993; 194: 784-790Crossref PubMed Scopus (46) Google Scholar, 29.van Rooijen J.J. Kamerling J.P. Vliegenthart J.F. Eur. J. Biochem. 1998; 256: 471-487Crossref PubMed Scopus (65) Google Scholar, 30.van Rooijen J.J. Voskamp A.F. Kamerling J.P. Vliegenthart J.F. Glycobiology. 1999; 9: 21-30Crossref PubMed Scopus (85) Google Scholar). One of our laboratories (15.Pak J. Pu Y. Zhang Z.T. Hasty D.L. Wu X.R. J. Biol. Chem. 2001; 276: 9924-9930Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) demonstrated that (i) hTHP is the main urinary protein binding specifically to type 1-fimbriated E. coli; (ii) removal of high mannose glycans from hTHP annuls this binding; and (iii) binding of the E. coli to uroplakin receptors is blocked by purified THP. These results suggest that by competing with urothelial receptors for type 1-fimbriated E. coli, urinary THP can prevent the E. coli from binding to the urothelial surface and that THP can serve as a major urinary defense factor against bacterial infections.In the present study, we reported several major species-specific differences in the chemical properties of THPs. We provided evidence that pTHP and hTHP differ significantly in their high mannose composition and that this difference results in their differential binding to the type 1-fimbriated E. coli. In addition, we demonstrated that pTHP and hTHP have different susceptibility to leukocyte elastase, thus providing an explanation for the different degrees of urinary degradation of the THPs. Finally, we showed that the species-specific expression of glycosyltransferases is responsible for different terminal sugar modifications of THPs in different species. These results document the species variations of the THPs and have functional implications on host adaptation to bacterial colonization and infections.EXPERIMENTAL PROCEDURESMaterials—Human urine was collected over 24 h from four healthy individuals. Bovine and pig urine was removed immediately postmortem from the bladders of two and three animals, respectively. Anti-serum to human THP was raised in rabbits as previously described (31.Bloomfield F.J. Dunstan D.R. Foster C.L. Serafini-Cessi F. Marshall R.D. Biochem. J. 1977; 164: 41-51Crossref PubMed Scopus (17) Google Scholar). Reduction/alkylation of THPs was performed as described by van Rooijen et al. (30.van Rooijen J.J. Voskamp A.F. Kamerling J.P. Vliegenthart J.F. Glycobiology. 1999; 9: 21-30Crossref PubMed Scopus (85) Google Scholar). Biotin-labeled concanavalin A (ConA), biotin-labeled Griffonia simplicifolia isolectin B4 (GS-IB4), anti-rabbit IgG conjugated with horseradish peroxidase, and pancreatic and leukocyte elastase were purchased from Sigma. Endoglycosidase H was from the Seikagaku Corporation. Glycopeptidase F (PNGase F) was from Roche Applied Science. Horseradish peroxidase-labeled streptavidin blocking reagent and ECL™ Western blotting reagent were from Amersham Biosciences, UK. [3H]KBH4 (67 mCi/mmol) and UDP[14C]Gal (325 Ci/mol) were from Amersham Biosciences. Bio-gel P-10 (fine) was from Bio-Rad. All other chemicals were of reagent grades. Modified glucose-free Eagle's medium was from Invitrogen.Bacterial Strains, Culture, and Metabolic Labeling—The P678-54 strain is a minicell-producing E. coli K12 derivative that expresses no fimbriae; J96 strain is a human pyelonephritis E. coli isolate expressing both type 1 and P fimbriae, whereas SH48 and HU849 are two recombinant strains derived by transfecting the nonfimbriated P678-54 strain with J96 genomic DNA fragments encoding the type 1 and P fimbriae (PapG1), respectively (32.O'Hanley P. Lark D. Falkow S. Schoolnik G. J. Clin. Invest. 1985; 75: 347-360Crossref PubMed Scopus (138) Google Scholar, 33.Stromberg N. Marklund B.I. Lund B. Ilver D. Hamers A. Gaastra W. Karlsson K.A. Normark S. EMBO J. 1990; 9: 2001-2010Crossref PubMed Scopus (218) Google Scholar, 34.Hull R.A. Gill R.E. Hsu P. Minshew B.H. Falkow S. Infect. Immun. 1981; 33: 933-938Crossref PubMed Google Scholar). KB96 and KB91 are two recombinant strains obtained by transfecting a fimH-null E. coli AAEC191A strain with fimH genes isolated from urinary tract infection or from the intestine, respectively; of these, the former strain expresses M1H fimbriae, and the latter M1L fimbrial variants (5.Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (300) Google Scholar). The fimbrial expression of clinical and recombinant E. coli strains was determined by yeast aggregation and hemagglutination as previously described (5.Sokurenko E.V. Chesnokova V. Dykhuizen D.E. Ofek I. Wu X.R. Krogfelt K.A. Struve C. Schembri M.A. Hasty D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8922-8926Crossref PubMed Scopus (300) Google Scholar, 9.Wu X.R. Sun T.T. Medina J.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9630-9635Crossref PubMed Scopus (261) Google Scholar). All strains were cultured in Lennox-Broth medium (Sigma) at 37 °C for 16 h, in methionine and cysteine-free Eagle's medium for 2 h, and then in Eagle's medium containing [35S]methionine and [35S]cysteine (PerkinElmer Life Sciences) for 2 h at room temperature. The labeled E. coli were washed four times in 0.02 m sodium phosphate buffer, pH 7.5, containing 0.14 m NaCl (PBS) and stored in PBS containing 30% glycerol at –80 °C until use.Purification of Urinary THPs and Preparation of THP Monomers— THPs were purified from pooled urine by either a diatomaceous earth filter (DEF) method (35.Serafini-Cessi F. Bellabarba G. Malagolini N. Dall'Olio F. J. Immunol. Methods. 1989; 120: 185-189Crossref PubMed Scopus (56) Google Scholar) or by the Tamm and Horsfall method (20.Tamm I. Horsfall F.L. Proc. Soc. Exp. Biol. Med. 1950; 74: 108-114Crossref Scopus (210) Google Scholar) with minor modifications. The urine was diluted with an equal volume of distilled water and brought to 0.58 m NaCl. After incubation at 4 °C for 16 h, the insoluble material was collected by centrifugation, resuspension in deionized water, and reprecipitation in the presence of 0.58 m NaCl. This step was repeated twice, and the final suspension was dialyzed exhaustively against deionized water. The monomeric forms of hTHP and pTHP were prepared by dissolving DEF-purified THPs (20 mg each) in phosphate buffer containing 8 m urea (pH 6.8) (36.Dawnay A. McLean C. Cattell W.R. Biochem. J. 1980; 185: 679-687Crossref PubMed Scopus (33) Google Scholar). The solutions were then extensively dialyzed against deionized water and lyophilized.Electrophoresis and Western Blotting—SDS-PAGE, at 8.5% or 10% acrylamide (see the figure legends), was performed as described (16.Cavallone D. Malagolini N. Serafini-Cessi F. Biochem. Biophys. Res. Commun. 2001; 280: 110-114Crossref PubMed Scopus (73) Google Scholar). When electrophoresis was performed in reducing conditions, β-mercaptoethanol was added to a final concentration of 1.5% (v/v). The proteins were either stained with Coomassie Brilliant Blue or transferred electrophoretically onto nitrocellulose membrane and probed with anti-THP antibodies followed by anti-rabbit IgG conjugated with horseradish peroxidase (16.Cavallone D. Malagolini N. Serafini-Cessi F. Biochem. Biophys. Res. Commun. 2001; 280: 110-114Crossref PubMed Scopus (73) Google Scholar). When the reactivity to lectins was analyzed, the blots were treated with 2.5% blocking reagent (Amersham Biosciences) and then incubated with biotinylated ConA lectin or with biotinylated GS-IB4 followed by streptavidin conjugated with horseradish peroxidase as previously described (37.Smilovich D. Malagolini N. Fagioli C. de Lalla C. Sitia R. Serafini-Cessi F. Glycobiology. 1998; 8: 841-848Crossref PubMed Scopus (13) Google Scholar). All of the blots were developed with ECL™ Western blotting reagent as recommended by the supplier.PNGase F and Elastase Digestion—Reduced/alkylated THPs were treated with PNGase F in 200 mm sodium phosphate buffer (pH 7.5) containing 50 mm EDTA, 0.1% Triton X-100 at 37 °C for 24 h. Alternatively, THPs were digested with pancreatic elastase or leukocyte elastase at an enzyme:protein ratio of 1:25 for 16 h as described by Jovine et al. (38.Jovine L. Qi H. Williams Z. Litscher E. Wassarman P.M. Nat. Cell Biol. 2002; 4: 457-461Crossref PubMed Scopus (264) Google Scholar).Preparation and Analysis of High Mannose Glycans—Approximately 35 mg of hTHP or pTHP were digested with Pronase (2 mg) at 60 °C for 48 h as previously described (39.Abbondanza A. Franceschi C. Licastro F. Serafini-Cessi F. Biochem. J. 1980; 187: 525-528Crossref PubMed Scopus (22) Google Scholar). The Pronase glycopeptides were fractionated on a column (1 × 80 cm) of Bio-Gel P10 (fine) equilibrated with 0.1 m NH4HCO3. The distribution of glycopeptides was identified by the phenol-sulfuric acid test (40.Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (39948) Google Scholar). The fractions corresponding to the elution position of high mannose glycopeptides were pooled, desalted on a Bio-Gel P2 column, and lyophilized. The high mannose glycopeptides were solubilized in 0.2 ml of 0.2 m sodium citrate buffer at pH 5 and subjected to endoglycosidase H treatment (27.Serafini-Cessi F. Dall'Olio F. Malagolini N. Biosci. Rep. 1984; 4: 269-274Crossref PubMed Scopus (47) Google Scholar). After reduction by [3H]KBH4, the oligosaccharides released were separated by HPLC with a water apparatus equipped with a LiChrosorb diol column eluted with 70:30 (v/v) acetonitrile:water. The 0.5-ml fractions were collected and counted for radioactivity.Assay of α1,3Gal-transferase from Human and Pig Kidney—Homogenates of outer medulla from human and pig kidney were obtained as previously described (16.Cavallone D. Malagolini N. Serafini-Cessi F. Biochem. Biophys. Res. Commun. 2001; 280: 110-114Crossref PubMed Scopus (73) Google Scholar). The assay mixture contained 0.1 m sodium cacodylate buffer, pH 6.0, 5 mm MnCl2, 0.5 mm UDP-[14C]Gal (5.6 dpm/pmol), 0.5% Triton X-100, 6.4 mm ATP, and 0.65 μmol of N-acetyllactosamine as an acceptor in a total volume of 50 μl and 70–80 μg of protein homogenate. Incubation was performed at 37 °C for 2 h and then stopped with 1 ml of cold water, and the mixture was passed through a column (1 × 3 cm) of Dowex 1 × 8 (Cl–) equilibrated with water. The column was eluted with 3 ml of water. The eluted sample was lyophilized and analyzed by HPLC as previously described (41.Malagolini N. Dall'Olio F. Guerrini S. Serafini-Cessi F. Glycoconj. J. 1994; 11: 89-95Crossref PubMed Scopus (16) Google Scholar). The fractions with the retention time of the trisaccharide (Galα1,3Galβ1,4GlcNAc) were counted for radioactivity.Solubility of THPs in Both Polymeric and Monomeric Forms—Purified THPs in polymeric and monomeric forms were dissolved in deionized water at a concentration of 0.4 mg/ml to obtain an optical density close to 0.400 at 277 nm. Each solution was divided into samples of 0.9 ml, to which 0.1 ml of a NaCl solution was added at increasing concentrations ranging from 0.4 to 1.6 m. In the control samples, 0.1 ml of deionized water was added. The samples were left at room temperature for 30 min and then centrifuged in a microcentrifuge (ALC International) for 30 min at 15,000 rpm, and the optical density of supernatants read at 277 nm.E. coli Binding Assay—The monomeric forms of hTHP and pTHP and BSA were dissolved in distilled water to different concentrations, and 100-μl aliquots were applied to a 96-well polystyrene microtiter plate. The solutions were left at room temperature for 30 min and at 4 °C for 16 h. After washing, the unoccupied sites were blocked with 3% BSA made in PBS and 0. 1% NaN3 for 2 h and then incubated for 2 h at room temperature with [35S]methionine/cysteine-labeled E. coli strains reconstituted in PBS containing 3% BSA and 0.1% NaN3 (5 × 105 cpm). After washing four times with PBS, the bound bacteria were dissolved in 1% SDS and quantified using a scintillation counter. All of the bindings were performed in triplicate.Analytical Methods—The protein concentration was determined by Lowry's method (42.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using BSA as a standard. Sugar content was determined by the phenol methods as described by Dubois et al. (40.Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (39948) Google Scholar).RESULTSDifference in N-linked Glycosylation Accounts for Different Electrophoretic Mobility of pTHP and hTHP—When total urinary proteins were resolved by SDS-PAGE under nonreducing conditions (Fig. 1A) and THPs were subsequently identified by Western blotting (Fig. 1B), pTHP clearly exhibited a faster electrophoretic mobility than that of hTHP and oxen THP. There were several minor, lower molecular weight bands in human urine that were also reactive with the THP antibody (Fig. 1B). These bands were, however, absent from pig urine, suggesting that hTHP is more prone to enzymatic degradation than pTHP (see below). Consistent with their urinary counterparts, pTHP purified either with the salt precipitation method of Tamm and Horsfall (20.Tamm I. Horsfall F.L. Proc. Soc. Exp. Biol. Med. 1950; 74: 108-114Crossref Scopus (210) Google Scholar) or with the diatomaceous earth retention method migrated faster than hTHP (Fig. 1C).Because one of the possibilities of different electrophoretic mobility of the glycoproteins may lie in the different degrees of glycosylation, we subjected both hTHP and pTHP to N-glycosidase (PNGase F) treatment. Under reducing conditions and prior to enzyme treatment, both pTHP and hTHP migrated more slowly than the nonreduced forms (Fig. 2), with pTHP migrating faster than hTHP. PNGase F treatment resulted in a dramatic decrease of the apparent molecular weights of both THPs and, more interestingly, abolished the difference of the electrophoretic mobility between the two THPs (Fig. 2). These data strongly suggest that pTHP and hTHP differ in N-linked glycosylation.Fig. 2Effect of deglycosylation on electrophoretic mobility of hTHP and pTHP. Reduced and alkylated THPs were incubated with PNGase F, electrophoresed in an 8.5% SDS-PAGE, and visualized either by Coomassie Blue staining (A) or by Western blotting using an anti-hTHP antibody (B). Note that deglycosylation abolished the difference of electrophoretic mobility between hTHP and pTHP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Different Susceptibility of hTHP and pTHP to Proteases—It has been recently shown that the N-terminal portion of hTHP (amino acids 1–291) is particularly susceptible to enzymatic degradation by pancreatic elastase (38.Jovine L. Qi H. Williams Z. Litscher E. Wassarman P.M. Nat. Cell Biol. 2002; 4: 457-461Crossref PubMed Scopus (264) Google Scholar). The fact that hTHP and pTHP exhibited different degrees of urinary degradation (Fig. 1B) prompted us to examine the relative susceptibility of purified THPs to enzymatic degradation. Fig. 3A shows that although hTHP was completely degraded by pancreatic elastase to a core peptide of approximately 48 kDa, pTHP was largely resistant. Because leukocytes are frequently present in human urine, we also subjected both THPs to leukocyte elastase treatment. Again, the bulk of hTHP was degraded, whereas the majority of pTHP was resistant to the enzyme digestion (Fig. 3B).Fig. 3Resistance of hTHP and pTHP to pancreatic or leukocyte elastase. Purified THPs in native forms were treated with pancreatic elastase (A) or with leukocyte elastase (B), followed by electrophoresis in a 10% SDS-PAGE in reducing conditions and Coomassie Blue staining. Note that pTHP is more resistant to the digestion by both enzymes than hTHP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reactivity of hTHP and pTHP to Lectins—The glycosylation type of hTHP and pTHP was examined by testing their reactivity with two different plant lectins: (i) ConA, which specifically recognizes the high mannose sequences; and (ii) GS-IB4, which recognizes the Galα1,3Gal sequence (43.Wood C. Kabat E.A. Murphy L.A. Goldstein I.J. Arch. Biochem. Biophys. 1979; 198: 1-11Crossref PubMed Scopus (143) Google Scholar). When the same amounts of pTHP and hTHP were used, ConA reacted with pTHP much more strongly than with hTHP (Fig. 4A; see below). A strong reactivity with GS-IB4 was also observed with pTHP, suggesting that the Galα1,3Galβ1,4GlcNAc epitope is present at the terminal nonreducing ends of pTHP polyantennary glycans. Consistent with this result, α1,3-galactosyltransferase, which is responsible for the assembly of Galα1,3Galβ1,4, was found to be highly expressed in the outer medulla of the pig kidney but not at all in the human kidney.Fig. 4Reactivity of THPs to plant lectins and detection of oligosaccharide transferase in the kidneys.A, equal amounts (15 μg) of pTHP and hTHP were resolved by SDS-PAGE, electrophoretically transferred to nitrocellulose membrane and probed with ConA or GS-IB4 as described in the text. Note the much stronger reaction of ConA and GS-IB4 to pTHP. B, the activity of α1,3Gal-transferase in the outer medulla (OM) of human and pig kidney was assayed. Note the strong activity of the enzyme in pig kidney but not in human kidney.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of High Mannose Glycans from THPs—The greater reactivity of ConA to pTHP than to hTHP prompted us to ascertain whether the two THPs differed in the high mannose glycans. 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