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Abstract
Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. Discussion/Conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.
Highlights
Inter-α-trypsin inhibitor (ITI) was first discovered as a serum proteinase inhibitor by Steinbuch and Loeb in 1961 [1]
A series of studies subsequently demonstrated ITI in the sera of a number of animals including, bovine, cervine, equine, porcine, donkey, lapine, canine, rat and ovine sources and its susceptibility to limited proteolysis by a trypsin-like serine proteinase yielding a number of characteristic trypsin inhibitory species [2,3,4,5]
Bikunin is unique among the proteoglycans in that its chondroitin sulfate (CS) chain acts as an internal linkage module for the attachment of a number of heavy chain proteins in ITI [5,13]
Summary
Inter-α-trypsin inhibitor (ITI) was first discovered as a serum proteinase inhibitor by Steinbuch and Loeb in 1961 [1]. Further studies led to the identification of two Kunitz protease inhibitor domains in the ITI light chain which was subsequently named bikunin [2,5]. An additional related large ITI serum inhibitor of 120 kDa was identified, which could be cleaved by limited digestion with kallikrein into 100 and 35 kDa fragments [6], this was named pre-α-trypsin inhibitor (pre-α-TI) [7]. Bikunin is unique among the proteoglycans in that its CS chain acts as an internal linkage module for the attachment of a number of heavy chain proteins in ITI [5,13]. Further studies have demonstrated up to six heavy chains attached to ITI in a number of tissues [14,15] and truncated heavy chains in fibrillated regions of osteoarthritic (OA) human articular cartilage (AC) [16]
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