SP220K is a novel matrix serine proteinase.

Matrix proteinases play a critical role in extracellular matrix remodeling, which is particularly involved in cancer invasion and metastasis. We have previously characterized and purified a new tetrameric serine proteinase (SP220K) from human kidney clear cell carcinoma plasma membranes. Here, we report that SP220K exhibits gelatinase activity as assessed both in solution and by zymography. Optimum gelatinase activity ranges between pH 7.5 to pH 9.0. Fibronectin and type I collagen were hydrolyzed by SP220K, at variance with laminin and type IV collagen. Like other trypsin-like fibronectin degrading proteinases, SP220K released the 29-kDa N-terminal heparin-binding domain of fibronectin. By using a panel of proteinase inhibitors, we found that the inhibition profile of SP220K was different from that of other known serine proteinases such as thrombin, trypsin, plasmin, plasminogen activators and tryptase. Altogether, our results indicate that SP220K corresponds to a novel matrix proteinase that exhibits a marked specificity for fibronectin and type I collagen.

Differential SP220K expression in renal carcinoma and oncocytoma cells.

SP220K is a newly described serine proteinase which displays guanidinobenzoatase activity in its inactive form and gelatinolytic activity in its active form. SP220K expression was studied in 20 renal clear-cell carcinomas and in a series of renal oncocytomas, a rare benign tumor derived from the kidney tubule epithelium. We provide evidence that SP220K expression, as assessed by guanidinobenzoatase activity, gelatin zymography and Western blot immunodetection, was increased markedly in cancer basolateral membranes compared to kidney cortex controls, whereas no signal was detectable in basolateral membranes from the 5 renal oncocytomas studied. Cytoplasms of carcinoma cells were immunodetected consistently, whereas no expression was seen in oncocytic cells from any of the oncocytomas studied (12/12). Endothelial cells were immunodetected in all 3 tissue types. Our data favor a potential mechanistic relationship between expression of the matrix proteinase SP220K and invasive phenotype in kidney epithelium proliferative processes.

Monomeric 55-kDa guanidinobenzoatase switches to a serine proteinase activity upon tetramerization. Tetrameric proteinase SP 220 K appears as the native form.

Guanidinobenzoatases are cell surface enzymes present in cells capable of migration or remodeling. The guanidinobenzoatase purified to homogeneity from human renal carcinoma did not display gelatinase activity under the 55-kDa form (Poustis-Delpont, C., Descomps, R., Auberger, P., Delque-Bayer, P., Sudaka, P., and Rossi, B. (1992) Cancer Res. 52, 3622-3628). We bring new insights into the structure-activity relationships of this enzyme using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]diisopropyl fluorophosphate labeling, gelatin zymography, and immunodetection using a polyclonal antibody raised against the 55-kDa entity. Upon aggregation into a 220-kDa form, the enzyme exhibited [3H]diisopropyl fluorophosphate labeling and diisopropyl fluorophosphate-inhibitable gelatinase activity whereas its capability to cleave p-nitrophenyl p'-guanidinobenzoate as a substrate was abolished. Thus, the guanidinobenzoatase property appears as a feature of a 55-kDa inactive form of a serine proteinase subunit. After boiling in the presence of sodium dodecyl sulfate (3% w/v), the 220-kDa entity subjected to SDS-polyacrylamide gel electrophoresis could be dissociated into a 55-kDa protein as shown by silver staining. The resulting 55-kDa band remained [3H]diisopropyl fluorophosphate-labeled and reacted with anti-55-kDa guanidinobenzoatase antibodies, strongly suggesting that the 220-kDa proteinase was a noncovalently associated tetramer. Interestingly, Triton X-100 extracts of renal carcinoma plasma membranes exhibited a 220-kDa serine proteinase activity, as expressed in gelatin zymography, which was barely detectable in the non-tumoral counterpart. It is noteworthy that an anti-55-kDa guanidinobenzoatase reactive 220-kDa species was also observed in renal carcinoma plasma membranes extracts as assessed by Western blot, whereas it was hardly visible in the non-tumoral counterpart. No signal was immunodetected at M(r) 55,000 in renal carcinoma and kidney cortex membranes in Western blot experiments. Taken together, our data support the idea that the enzyme is expressed under its tetrameric form in the membrane. The purified enzyme is able to exhibit a serine proteinase activity when it recovers its native tetrameric form. This high molecular weight tetrameric proteinase SP 220 K appears as a new member of the cell surface serine protease family.