Bait region cleavage and complex formation of human alpha2M with a Porphyromonas gingivalis W50 protease is not accompanied by enzyme inhibition.
Three isoforms of extracellular Arg-specific proteases of P. gingivalis, W50, HRgpA, RgpAcat and mt-RgpAcat, which are all products of the same gene, show identical enzymatic properties toward small chromogenic substrates but have different subunit organisation and molecular size. In order to examine the potential inhibition of these proteases in vivo by host protease inhibitors, the interaction of HRgpA (approximately 110 kDa) and RgpAcat (approximately 55 kDa) with human (alpha2M and their cytotoxicity toward cultured fibroblasts were investigated. Both enzymes formed complexes with (alpha2M as shown by gel filtration chromatography and both cleaved the 'bait' region at Arg696-Leu697. However, whereas (alpha2M-RgpAcat) complex was unable to hydrolyse large substrates such as hide powder azure, (alpha2M-HRgpA) complex hydrolysed both small and large substrates. HRgpA was able to bind to alpha2M saturated with trypsin and also to methylamine-treated alpha2M. This suggested that HRgpA is able to bind to both 'slow' and 'fast' forms of alpha2M and formation of (alpha2M:HRgpA) complex does not trap HRgpA and cause inhibition of activity toward hide powder azure. However, the (alpha2M-HRgpA) complex is not able to cleave other alpha2M molecules, which suggests that the active site of HRgpA in the complex is constrained probably due to steric reasons. The (alpha2M-HRgpA) complex was cytotoxic to 3T3 cells, causing them to round up and detach from the surface with a reduction in metabolic activity.
Rangarajan
M
M
Department of Oral Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, UK.
Scragg
M A
MA
Curtis
M A
MA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D008744:Methylamines / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D008744:Methylamines; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; C027451:methylamine; D010450:Endopeptidases