Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity.
Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.
Raymond
Wilfred W
WW
Cardiovascular Research Institute, University of California at San Francisco, San Francisco, CA 94143, USA.
Su
Sharon
S
Makarova
Anastasia
A
Wilson
Todd M
TM
Carter
Melody C
MC
Metcalfe
Dean D
DD
Caughey
George H
GH
D000328:Adult; D000804:Angiotensin II / Q000096:biosynthesis*; D002648:Child; D053818:Chymases / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D008407:Mast Cells / Q000201:enzymology* / Q000378:metabolism; D008415:Mastocytosis / Q000097:blood / Q000201:enzymology; D010865:Pilot Projects; D011485:Protein Binding; D011994:Recombinant Proteins / Q000097:blood / Q000378:metabolism; D044967:Serum / Q000201:enzymology*; D013379:Substrate Specificity; D000511:alpha-Macroglobulins / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; D000804:Angiotensin II; C509226:CMA1 protein, human; D053818:Chymases