Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting

Abstract

Biotinylated trypsin (bT) was used as a probe on Western blots of 10-20% polyacrylamide gradient Tris-Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S--300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of approximately 6 kDa was also detectable in cartilage extracts after prolonged storage at 4 degrees C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight 'native' cartilage SPI was distinct from ovine alpha1-proteinase inhibitor (alpha1-PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)-PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.

Affinity and Western blotting reveal homologies between ovine intervertebral disc serine proteinase inhibitory proteins and bovine pancreatic trypsin inhibitor

“…We have previously characterised 6-12 and 58 kDa ovine cartilage SPIs which also cross react with the chicken anti-BPTI antibody we have used in this study [13,18,19]. The 6-12 kDa serine proteinase inhibitor is a highly cationic protein (pI .10.5) while the 58 kDa serine proteinase inhibitor is anionic (pI *4.5) and apparently represents a precursor form of the smaller SPI [18,19].…”

“…The 6-12 kDa serine proteinase inhibitor is a highly cationic protein (pI .10.5) while the 58 kDa serine proteinase inhibitor is anionic (pI *4.5) and apparently represents a precursor form of the smaller SPI [18,19]. Chymotrypsin affinity chromatography of the 58 kDa SPI eventually converted it into the 12 kDa form, however a 36 kDa intermediate form was also occasionally observed during this procedure and has also been observed in some stored preparations of the 58 kDa SPI [19]. The 34-36 Da SPIs we have encountered in this study and which are also present in canine IVD specimens may therefore represent differentially processed forms of a larger precursor SPI, while the smaller *12 kDa species may represent the end product [18,19].…”

“…Chymotrypsin affinity chromatography of the 58 kDa SPI eventually converted it into the 12 kDa form, however a 36 kDa intermediate form was also occasionally observed during this procedure and has also been observed in some stored preparations of the 58 kDa SPI [19]. The 34-36 Da SPIs we have encountered in this study and which are also present in canine IVD specimens may therefore represent differentially processed forms of a larger precursor SPI, while the smaller *12 kDa species may represent the end product [18,19]. The fact that a range of inhibitory species are detected using biotinylated trypsin by Affinity blotting which only detects biologically active SPI species apparently indicates that the SPIs are stable to the proteolytic processing they have received in vivo.…”

A Retrospective Analysis of the Cartilage and Intervertebral Disc Kunitz Protease Inhibitory Proteins Identifies These as Members of The Inter-α-Trypsin Inhibitor Superfamily with potential roles in the protection of the articulatory surface

“…In the course of our studies on the SPIs of AC and IVD we have developed a sensitive Affinity blotting procedure using biotinylated trypsin (bT) which can be used alongside conventional antibody blots. This is a useful sensitive technique capable of detecting low ng-pg levels of SPI and also demonstrates that the SPI is in a biologically active form (21,23,24). In the present study we have used both of these techniques which complement one another to demonstrate that many of the SPIs previously identified in AC and IVD are in fact members of the ITI superfamily (18,19,21,(25)(26)(27)(28)(29)(30)(31).Since its discovery in 1961 as a serum protease inhibitor the precise functional role of ITI have been a bit of a mystery.…”

“…This is a useful sensitive technique capable of detecting low ng-pg levels of SPI and also demonstrates that the SPI is in a biologically active form (21,23,24). In the present study we have used both of these techniques which complement one another to demonstrate that many of the SPIs previously identified in AC and IVD are in fact members of the ITI superfamily (18,19,21,(25)(26)(27)(28)(29)(30)(31).Since its discovery in 1961 as a serum protease inhibitor the precise functional role of ITI have been a bit of a mystery. Despite displaying potent protease inhibitory properties against a number of serine proteases including leucocyte elastase and cathepsin G, pancreatic trypsin and chymotrypsin, plasmin, kallikrein and some of the coagulation cascade proteinases the requirement for additional serine protease inhibitory activity in serum was questionable given that serum already contained 1-proteinase inhibitor, antithrombin-III, C1 esterase inhibitor, 2-antiplasmin, 1-antichymotrypsin, and 2-macroglobulin which display a broad inhibitory spectrum and a high capacity for protease inhibition.…”