Down-regulation of secretion of human complement component C2 by the product of an alternatively spliced C2 messenger RNA.
We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.
Tsukamoto
H
H
Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294, USA.
Tousson
A
A
Marchase
R B
RB
Volanakis
J E
JE
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D020126:Brefeldin A; D002451:Cell Compartmentation / Q000187:drug effects; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003175:Complement C2 / Q000235:genetics / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D015536:Down-Regulation; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics
D003175:Complement C2; D003517:Cyclopentanes; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D020126:Brefeldin A