Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1.
Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110 kDa in human milk, which contained no HAI-1 and resisted dissociation in boiling SDS in the absence of reducing agents. These complexes were further purified and dissociated into 80-kDa and 45-kDa fragments by treatment with reducing agents. Proteomic and immunological methods identified the 45-kDa fragment as the noncatalytic domains of matriptase and the 80-kDa fragment as the matriptase serine protease domain covalently linked to one of three different secreted serpin inhibitors: antithrombin III, alpha1-antitrypsin, and alpha2-antiplasmin. Identification of matriptase-serpin inhibitor complexes provides evidence for the first time that the proteolytic activity of matriptase, from those cells that express no or low levels of HAI-1, may be controlled by secreted serpins.
Greenebaum Cancer Ctr., Dept. of Biochemistry and Molecular Biology, Univ. of Maryland Baltimore, BRB 10-027, 655 W. Baltimore St., Baltimore, MD 21201, USA.
I-Chu
IC
Tseng
Feng-Pai
FP
Chou
Sheng-Feng
SF
Su
Michael
M
Oberst
Nandakumar
N
Madayiputhiya
Ming-Shyue
MS
Lee
Jehng-Kang
JK
Wang
David E
DE
Sloane
Michael
M
Johnson
Chen-Yong
CY
Lin
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D008895:Milk, Human / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D053491:Proteinase Inhibitory Proteins, Secretory / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D015843:Serpins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D000979:alpha-2-Antiplasmin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism
D053491:Proteinase Inhibitory Proteins, Secretory; C109149:SPINT1 protein, human; D015843:Serpins; D000515:alpha 1-Antitrypsin; D000979:alpha-2-Antiplasmin; D000990:Antithrombin III; D012697:Serine Endopeptidases; C485596:ST14 protein, human