Significance of prohormone convertase 2, PC2, mediated initial cleavage at the proglucagon interdomain site, Lys70-Arg71, to generate glucagon.
To define the biological significance of the initial cleavage at the proglucagon (PG) interdomain site, K70-R71 downward arrow, we created two interdomain mutants, K70Q-R71Q and R71A. Cotransfection studies in GH4C1 cells show significant amounts of glucagon production by PC2 along with some glicentin, glicentin-related polypeptide-glucagon (GRPP-glucagon) and oxyntomodulin from wild-type PG. In contrast, a larger peptide, PG 33-158, and low amounts of GRPP-glucagon are predominantly generated from interdomain mutants. HPLC analysis shows a 5-fold increase in glucagon production by PC2 from wild-type PG and a corresponding 4-fold lower accumulation and secretion of unprocessed precursor relative to interdomain mutants. PC2 generates significant levels of glucagon from a glicentin (PG 1-69) expression plasmid, whereas PC1/3 produces only modest amounts of oxyntomodulin. Employing a major PG fragment (PG 72-158) expression plasmid, we show that PC1/3 predominantly generates glucagon-like peptide (GLP)-1, whereas PC2 produces only N-terminally extended GLP-1. Surprisingly, production of GLP-1 and GLP-2 by PC1/3 from interdomain mutants, compared with wild-type PG, is not significantly impaired. In addition to PC2 and PC1/3, PC5/6A and furin are also able to cleave the sites, K70-R71 downward arrow and R107-X-R-R110 downward arrow in PG. We show a much greater ability of furin to cleave the monobasic site, R77 downward arrow, than at the dibasic site, R124-R125 downward arrow, which is also weakly processed by PC5/6A, indicating overlapping specificities of these two convertases mainly with PC1/3. We propose here a trimer-like model of the spatial organization of the hormonal sequences within the PG molecule in which the accessibility to prohormone convertase action of most cleavage sites is restricted with the exception of the interdomain site, K70-R71, which is maximally accessible.
Dey
Arunangsu
A
Department of Biochemistry and Molecular Biology, Uni-versity of Chicago, Chicago, Illinois 60637, USA.
Lipkind
Gregory M
GM
Rouillé
Yves
Y
Norrbom
Christina
C
Stein
Jeffrey
J
Zhang
Chunling
C
Carroll
Raymond
R
Steiner
Donald F
DF
D000818:Animals; D006224:Cricetinae; D005934:Glucagon / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1; D053767:Glucagon-Like Peptide 2; D008958:Models, Molecular; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology; D052336:Proglucagon; D045706:Proprotein Convertase 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D053767:Glucagon-Like Peptide 2; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D052336:Proglucagon; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D045706:Proprotein Convertase 2