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

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, porci...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2008-12, Vol.144 (6), p.725-732
Main Authors: Telugu, Bhanu Prakash V.L, Green, Jonathan A
Format: Article
Language:English
Subjects:
Animals
Aspartic Acid Endopeptidases - genetics
Aspartic Acid Endopeptidases - isolation & purification
Aspartic Acid Endopeptidases - metabolism
aspartic peptidases
Evolution, Molecular
Female
Hydrogen-Ion Concentration
Kinetics
Phylogeny
placenta
porcine
Pregnancy
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Swine - metabolism
trophoblast
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Description
Summary: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.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvn127