Pancreatic trypsin activates human promatrix metalloproteinase-2.
In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.
Lindstad
Rune I
RI
Department of Biochemistry, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway.
Sylte
Ingebrigt
I
Mikalsen
Svein-Ole
SO
Seglen
Per O
PO
Berg
Eli
E
Winberg
Jan-Olof
JO
D000477:Alkylating Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology* / Q000378:metabolism; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D017186:Titrimetry; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*
D000477:Alkylating Agents; D003902:Detergents; D004792:Enzyme Precursors; D007527:Isoenzymes; D019715:Tissue Inhibitor of Metalloproteinase-1; D000077423:Polidocanol; D011092:Polyethylene Glycols; D014108:Tosylphenylalanyl Chloromethyl Ketone; D014357:Trypsin; D020778:Matrix Metalloproteinase 2; D002118:Calcium