The major human urinary trypsin inhibitor is a proteoglycan

Abstract

The major urinary trypsin inhibitor (MI 44000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride.This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (MI 26000) but still inhibits trypsin.Several acid-resistant trypsin inhibitors with molecular masses, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE), ranging from 40 kDa -45 kDa, have been isolated from human urine [l -41. All these inhibitors show an immunological cross-reactivity with intera-trypsin inhibitor.Hochstrasser et al.[5] first described a physiological urinary inhibitor having an apparent molecular mass of 34 kDa as calculated from its specific activity. This inhibitor (HI-30) has an apparent molecular mass of 30 kDa, as estimated by gel chromatography [6], but 40 kDa in SDS-PAGE [4].We previously described the isolation and characterization of a human urinary trypsin inhibitor (UTI) with the same aminoterminal sequence as 81. This sequence was recently found to be identical with that of inter-a-trypsin inhibitor [9, 101; those data tend to confirm the hypothesis that the different urinary trypsin inhibitors isolated so far are derived from inter-a-trypsin inhibitor.The proportion and nature of the carbohydrate reported for those inhibitors is variable, ranging from 10% to 50% [l,3, 71. According to Hochstrasser et al. [ll] the carbohydrate content of HI-30 accounts for 50% of the total weight and includes two carbohydrate chains: one is attached N-glycosidically via an asparaginyl residue, the other is 0-glycosidically linked to the polypeptide via a seryl residue in position 10. This last chain is made up of at least 48 Nacetylgalactosamine, 2 glucose, 18 galactose and 2 N-acetylneuraminic acid residues [6]. Such a composition is quite unusual for a glycoprotein.In this report we describe a slightly modified procedure for isolation of urinary trypsin inhibitor and we present evidence that it consists a proteoglycan. MATERIALS AND METHODSSephacryl S-200, DEAE-Sephacel and ConA-Sepharose were purchased from Pharmacia, DEAE-Trisacryl was from IBF. Acrylamide and N-N'-methylenebisacrylamide were from BDH, agarose (Indubiose A 37) from IBF. Bovine pancreatic trypsin (treated with Tos-Phe-CH2C1) and L-benzoylarginine p-nitroanilide were purchased from Merck, chondroitinase ABC (from Proteus vulgaris) and bovine testicular hyaluronidase were from ...

The Uniform Galactose 4‐Sulfate Structure in the Carbohydrate‐Protein Linkage Region of Human Urinary Trypsin Inhibitor

“…The structural uniformity in the linkage hexasaccharide structure of IT1 and UTI is in marked contrast to the heterogeneity demonstrated in the linkage hexasaccharides isolated from cartilaginous chondroitin sulfate whose linkage regions are sometimes but not always phosphorylated on the Xyl residue or sulfated on the Gal residue(s). The uniform structure containing the novel 4-sulfated Gal residue in the linkage region of UTI and IT1 may imply its significance in the biosynthetic mechanism of chondroitin sulfate.Keywords: chondroitin sulfate; 'H-NMR; inter-a-trypsin inhibitor; sulfated oligosaccharides; urinary trypsin inhibitor.Urinary trypsin inhibitor (UTI) is a serine-protease inhibitor present in human urine and is a proteoglycan bearing an undersulfated chondroitin 4-sulfate chain (Balduyck et al, 1986: Toyoda et al, 1993. The purified UTI has been used therapeutically in patients with rheumatoid arthritis.…”

“…Urinary trypsin inhibitor (UTI) is a serine-protease inhibitor present in human urine and is a proteoglycan bearing an undersulfated chondroitin 4-sulfate chain (Balduyck et al, 1986: Toyoda et al, 1993. The purified UTI has been used therapeutically in patients with rheumatoid arthritis.…”

“…This type of linkage is callcd a proteinglycosaminoglycan-protein (PGP) cross-link (Enghild et al, 1991). In biosynthesis, the chondroitin 4-sulfate chain is synthesized on and attached to a serine residue of the L chain through the conventional carbohydrate-protein linkage structure GlcAp1-3Gal~1-3Gal~l-4Xyl~l-O-Ser (where GlcA represents D-gIUCUronic acid; Balduyck et al, 1986;Enghild et al, 1993). The H, chain is bound to the chondroitin 4-sulfate chain through a unique ester bond between the a-carboxylate of the COOH-terminal Asp and C6 OH of an internal GalNAc of the chondroitin 4-sulfate chain (Enghild et al, 1993), and the H, chain has also been demonstrated to be linked to the chondroitin 4-sulfate with similar architecture (Morelle et al, 1994).…”

Chondroitin sulphate covalently cross‐links the three polypeptide chains of inter‐α‐trypsin inhibitor

“…They were further eliminated by anion-exchange chromatography. In agreement with the low sulphate content of the chondroitin sulphate chain [17], the GAG-containing material was eluted from the monoQ anion-exchange column at a lower salt concentration than chondroitin 4-sulphate, used as marker. The amino acid and carbohydrate compositions as well as the sequence of this component, homogeneous by RP-HPLC analysis, were in agreement with the expected structures for the IT1 fragment previously described (Fig.…”

One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate

“…UTI has a molecular mass of 4045 kDa (Tanaka et al, 1982), which is decreased to 26 kDa upon chondroitinase ABC treatment (Balduyck et al, 1986). It contains two sulphate groups and apparently about 20 GluA-GalNAc repeats (Balduyck et al, 1986;Selloum et al, 1987). No …”