The human homologue of the Escherichia coli htrA gene product was identified by the differential display analysis of transcripts expressed in osteoarthritic cartilage. This transcript was identified previously as being repressed in SV40-transformed fibroblasts (Zumbrunn, J., and Trueb, B. (1996) FEBS Lett. 398, 187-192). Levels of HtrA mRNA were elevated ϳ7-fold in cartilage from individuals with osteoarthritis compared with nonarthritic controls. Differential expression of human HtrA protein was confirmed by an immunoblot analysis of cartilage extracts. Human HtrA protein expressed in heterologous systems was secreted and exhibited endoproteolytic activity, including autocatalytic cleavage. Conversion by mutagenesis of the putative active site serine 328 to alanine eliminated the enzymatic activity. Serine 328 was also found to be required for the formation of a stable complex with ␣ 1 -antitrypsin. We have determined that the HtrA gene is highly conserved among mammalian species: the amino acid sequences encoded by HtrA cDNA clones from cow, rabbit, and guinea pig are 98% identical to human. In E. coli, a functional htrA gene product is required for cell survival after heat shock or oxidative stress; its role appears to be the degradation of denatured proteins. We propose that mammalian HtrA, with the addition of a new functionality during evolution, i.e. a mac25 homology domain, plays an important role in cell growth regulation.

Lysosomal glycosylasparaginase is encoded as a 36.5 kDa polypeptide that is post‐translationally processed to subunits of 19.5 kDa (heavy) and 15 kDa (light). Recombinant glycosylasparaginase has been expressed in Spodoptera frugiperda insect cells enabling the precursor and processed forms to be isolated and their catalytic potential determined. Only the subunit conformation was functional indicating glyeosylasparaginase is encoded as an inactive zymogen. The newly created amino terminal residue of the light subunit following maturation, Thr‐206, is believed to be involved in the catalytic mechanism [1992, J. Biol. Chem. 267,6855‐6858]. Here we have constructed two amino acid substitution mutants replacing Thr‐206 with Ala‐206 or Ser‐206 and demonstrate that both destroy enzyme activity.

We have identified a splice variant of human neutrophil collagenase (MMP-8) transcript (MMP-8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP-8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP-8, the translation product of MMP-8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP-8alt protein may lead to uncover novel roles it plays in cellular physiology.z 1999 Federation of European Biochemical Societies.