Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis.
Insulin-like growth factor-2 (IGF-2) is expressed in most embryonic tissues and is required for normal development during gestation. After birth IGF-2 expression is extinguished in most tissues, but the gene is often reactivated during tumorigenesis. Tumors secrete high molecular weight forms of IGF-2 that result from aberrant post-translational processing of pro-IGF-2. As a first step toward understanding how high molecular weight IGF-2 peptides might contribute to tumor progression, we have characterized the biosynthesis of IGF-2 in a human embryonic cell line. We have found that pro-IGF-2 can initially form two disulfide isomers that undergo rearrangement to a single conformation in vivo. The addition of N-acetylgalactosamine to Ser71, Thr72, Thr75, and Thr139 likely occurs in the cis- Golgi apparatus. Sialic acid addition begins in the trans- Golgi apparatus, but IGF-2 peptides must reach the trans-Golgi network for oligosaccharide maturation to be completed. Endoproteolysis occurs concomitant to or slightly after oligosaccharide maturation. Cleavage was observed only at Arg104, resulting in the secretion of IGF-2-(1-104) and free E-peptide. Proteolysis required basic residues in the P1 (Arg104) and P4 (Arg101) positions, was completely blocked by a furin inhibitor, and was enhanced by coexpression with furin, PACE4, PC6A, PC6B, and LPC. These data suggest that members of the subtilisin-related proprotein convertase family mediate processing of pro-IGF-2 at Arg104. We did not detect the IGF-2 peptides that are most abundant in normal serum, mature IGF-2, and IGF-2-(1-87), in this expression system, which indicates that novel endoproteases are responsible for generating these products.
Duguay
S J
SJ
Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA.
Jin
Y
Y
Stein
J
J
Duguay
A N
AN
Gardner
P
P
Steiner
D F
DF
D001120:Arginine / Q000378:metabolism; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism*; D006031:Glycosylation; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000378:metabolism; D014162:Transfection
D006133:Growth Substances; D011498:Protein Precursors; C066038:proinsulin-like growth factor II; D007335:Insulin-Like Growth Factor II; D001120:Arginine; D010450:Endopeptidases; D013381:Subtilisins