Complex formation between human kallikrein 13 and serum protease inhibitors.
BACKGROUND
The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein gene 13 (KLK13) is a member of this family and encodes for a trypsin-like, secreted serine protease (hK13). Given that other kallikreins are sequestered by serum protease inhibitors, we hypothesized that hK13 may also interact with similar inhibitors. Our objective was to identify serum protease inhibitors that interact with human hK13.


METHODS
Recombinant hK13 produced in yeast was added to male and female sera and various biological fluids and the spiked samples were analyzed with an hK13 ELISA assay. Enzymatically active hK13 was 125I-labeled and used in in vitro reactions with candidate protease inhibitors and serum samples. The mixtures were then subjected to gel filtration and SDS-PAGE analysis. Candidate inhibitors were also tested in enzymatic assays of hK13 activity.


RESULTS
The recovery of recombinant hK13 from male and female sera, measured by three versions of the hK13-ELISA, ranged from 5% to 10%. The same recovery was obtained when serum samples from males and females were spiked with hK13 from amniotic fluid and seminal plasma. However, when hK13 was added to other biological fluids, such as amniotic fluid and breast milk, recovery ranged from 70% to 98%. In vitro analysis indicated that enzymatically active 125I-labeled hK13 forms SDS-stable complexes with alpha2-antiplasmin, alpha2-macroglobulin and alpha1-antichymotrypsin. When added to serum, active hK13 formed stable complexes with molecular masses corresponding to hK13 and the inhibitors mentioned above.


CONCLUSIONS
hK13 interacts and forms complexes with serum protease inhibitors, including alpha2-macroglobulin, alpha1-antichymotrypsin and alpha2-antiplasmin.
Kapadia
Carl
C
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.
Yousef
George M
GM
Mellati
Ali Awsat
AA
Magklara
Angeliki
A
Wasney
Gregory A
GA
Diamandis
Eleftherios P
EP
D000818:Animals; D001202:Ascitic Fluid / Q000737:chemistry; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D007610:Kallikreins / Q000037:antagonists & inhibitors* / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010051:Ovarian Neoplasms / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012661:Semen / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C470778:KLK13 protein, human; D007610:Kallikreins; D014357:Trypsin