Expression, purification, characterization, and x-ray analysis of selenomethionine 215 variant of leukocyte collagenase

Pieper M, Betz M, Budisa N, GomisRuth FX, Bode W, Tschesche H. Expression, purification, characterization, and x-ray analysis of selenomethionine 215 variant of leukocyte collagenase. JOURNAL OF PROTEIN CHEMISTRY. 1997;16(6):637-650.Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et al. (1993) FEBS 331, 134-140; Stocker et al. (1995) Protein Sci. 4, 823-840]. The distal S-CH3 moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by selenomethionine, Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry, The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in changed kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon urea denaturation, The X-ray crystal structure of this selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site

Collagenase specificity in chondrosarcoma metastasis

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection