Expression and complement d activity of porcine adipsin.
To learn how signals from adipocytes might be involved in regulation of energy intake and storage, we have begun to characterize the porcine complement protein, adipsin. Adipsin was originally identified as a protein that is produced rather specifically by adipocytes, is secreted, and is nearly absent in several obese rodent models. We now report that porcine adipsin mRNA sequence is 74% identical to rat and predicts a protein that has 82 and 68% identity to human and rat forms, respectively. Porcine adipsin has none of the asparagine glycosylation consensus sites which make recombinant expression of mouse adipsin in Escherichia coli impractical. We present a method for engineering the porcine cDNA to facilitate expression by E. coli and provide a protocol for refolding and purifying porcine adipsin protein and for immunoassay. We have found that in addition to adipose tissue, adipsin mRNA is present in gut tissues. Coupled with the fact that adipsin is required for processing of complement C3a-desArg, and that C3a-desArg is a potent stimulant of fatty acid acylation in adipocytes, the production of adipsin in the gut may be related to a mechanism for adipocyte removal of lipid from chylomicrons.
Miner
J L
JL
Monsanto Company, Lincoln, Nebraska 68583-0908, USA. jminer1@unl.edu
Hahn
K J
KJ
Spurlock
M E
ME
Staten
N R
NR
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods*; D011416:Complement Factor D / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013552:Swine; D014018:Tissue Distribution
D012333:RNA, Messenger; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C493488:CFD protein, human; D011416:Complement Factor D; C493491:complement factor D, mouse