Differential processing of proenkephalin by prohormone convertases 1(3) and 2 and furin.
Recombinant vaccinia virus vectors were used to coexpress mouse prohormone convertase 1 (mPC1), mPC2, or human furin together with human proenkephalin in GH4C1 cells (rat pituitary somatomammotrophs) to examine the proteolytic processing of proenkephalin by these enzymes. Radioimmunoassays performed on high pressure gel permeation size-fractionated extracts obtained from GH4C1 cells and corresponding conditioned media revealed distinct profiles of immunoreactivity for products generated by each enzyme. PC1 produced intermediate sized processing products (3-10 kDa); the major immunoreactive enkephalin-containing species observed eluted at the positions of peptide B, the 5.3-kDa fragment, and free Leu5-enkephalin. PC2 exhibited a more complete processing profile. The major immunoreactive enkephalins produced were free Met5-enkephalin-Arg-Phe, free Met5-enkephalin-Arg-Gly-Leu, free Leu5-enkephalin, and free Met5-enkephalin. Thus PC2 appears to be more capable of generating active opioid units from proenkephalin than is PC1. Finally, furin cleaved proenkephalin to generate peptide B, an unidentified peak between the 18- and 5.3-kDa fragments, and a small amount of the 5.3-kDa fragment. Radiosequencing data verified that the production of the 5.3-kDa fragment by PC1 occurred as a result of a Lys-Lys cleavage. The ability of PC1 to cleave proenkephalin (but not proopiomelanocortin) at a Lys-Lys site implies that the structural context of the paired basic cleavage site may be more important in the determination of cleavage specificity than the particular pair of basic residues at the site.
Breslin
M B
MB
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Lindberg
I
I
Benjannet
S
S
Mathis
J P
JP
Lazure
C
C
Seidah
N G
NG
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D004745:Enkephalins / Q000378:metabolism*; D045683:Furin; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism*
D017077:Culture Media, Conditioned; D004745:Enkephalins; D011498:Protein Precursors; D011994:Recombinant Proteins; C029992:proenkephalin; D043484:Proprotein Convertases; D013381:Subtilisins; D045683:Furin; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; C473668:Pcsk2 protein, mouse; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases