Processing of mouse proglucagon by recombinant prohormone convertase 1 and immunopurified prohormone convertase 2 in vitro.
The mouse tumor cell line alpha TC1-6 was used as a model system to examine the post-translational processing of proglucagon. Determination of the mouse preproglucagon cDNA sequence and comparison with the published sequences of rat and human preproglucagons revealed nucleic acid homologies of 89.1 and 84%, respectively, and amino acid homologies of 94 and 89.4%, respectively. Immunohistochemical analyses with antibodies directed against PC2 and glucagon colocalized both the enzyme and substrate within the same secretory granules. PC1 was also immunolocalized in secretory granules. Cells were metabolically labeled with [3H]tryptophan, and extracts were analyzed by reverse-phase high pressure liquid chromatography. Radioactive peptides with retention times identical to those of synthetic peptide standards were recovered and subjected to peptide mapping to verify their identities. To determine the potential role of PC1 and PC2 in proglucagon processing, 3H-labeled proglucagon was incubated in vitro with recombinant PC1 and/or immunopurified PC2. Both enzymes cleaved proglucagon to yield the major proglucagon fragment, glicentin, and oxyntomodulin, whereas only PC1 released glucagon-like peptide-I from the major proglucagon fragment. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. These results suggest a potential role for PC1 and/or PC2 in cleaving several of the normal products, excluding glucagon, from the mouse proglucagon precursor.
Rothenberg
M E
ME
Department of Anatomy and Cell Biology, Emory University, Atlanta, Georgia 30322, USA.
Eilertson
C D
CD
Klein
K
K
Zhou
Y
Y
Lindberg
I
I
McDonald
J K
JK
Mackin
R B
RB
Noe
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D005934:Glucagon / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D052336:Proglucagon; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013381:Subtilisins / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007106:Immune Sera; D011498:Protein Precursors; D011994:Recombinant Proteins; D052336:Proglucagon; D005934:Glucagon; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases