N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells.
Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.
Ladenheim
R G
RG
Unité de Génétique et Biochimie du Développement, Institut Pasteur, Paris, France.
Seidah
N G
NG
Rougeon
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004792:Enzyme Precursors / Q000235:genetics*; D006026:Glycoside Hydrolases; D006031:Glycosylation; D007700:Kinetics; D046911:Macromolecular Substances; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010911:Pituitary Neoplasms; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012083:Renin / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013363:Submandibular Gland / Q000201:enzymology*; D014162:Transfection
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D012083:Renin