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Journal of Biochemistry Advance Access originally published online on October 3, 2008
Journal of Biochemistry 2008 144(6):725-732; doi:10.1093/jb/mvn127
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Characterization of the Peptidase Activity of Recombinant Porcine Pregnancy-associated Glycoprotein-2

Bhanu Prakash V.L. Telugu and Jonathan A. Green*

Division of Animal Sciences, University of Missouri, 163 ASRC, Columbia, MO 65211, USA

*To whom correspondence should be addressed. Tel: +573-884-1697, Fax: +573-882-6827, E-mail: greenjo{at}missouri.edu

Received June 8, 2008; Accepted September 15, 2008


  Abstract

The pregnancy-associated glycoproteins (PAGs) belong to the aspartic peptidase family. They are expressed exclusively in trophoblasts of even-toed ungulates such as swine, cattle, sheep, etc. In pigs, two distinct PAG transcripts (and some variants) have been described. One of the transcripts, porcine PAG-1 (poPAG-1) may not be capable of acting as a peptidase. The second transcript, poPAG-2, possesses a conserved catalytic centre and has been predicted, but not shown, to have proteolytic activity. The thrust of this work was to test such a possibility. PoPAG-2 was expressed as a recombinant protein with an amino-terminal ‘FLAG-tag’ in a Baculoviral expression system. The expressed proteins were affinity purified by using an anti-FLAG antibody. The purified preparations were then analysed for proteolytic activity against a fluorescent substrate. Porcine PAG-2 had optimal proteolytic activity around pH 3.5. Against this substrate, it had a kcat/Km of 1.2 µM–1 s–1 and was inhibited by the aspartic peptidase inhibitor, pepstatin A, with a Ki of 12.5 nM. Since the proteolytic activity of PAGs in the pig has now been established, the search for putative substrates to gain insight into the physiological role of PAGs will likely be the focus of future investigations.

Key Words: aspartic peptidases, placenta, porcine, pregnancy, trophoblast

Abbreviations: PAG, pregnancy-associated glycoprotein; poPAG, porcine pregnancy-associated glycoprotein; pepF, pepsinogen F; AP, aspartic peptidase


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