Assembly and secretion of fibrinogen. Degradation of individual chains.
Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.
Roy
S
S
Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021.
Yu
S
S
Banerjee
D
D
Overton
O
O
Mukhopadhyay
G
G
Oddoux
C
C
Grieninger
G
G
Redman
C
C
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002645:Chickens; D002738:Chloroquine / Q000494:pharmacology; D005340:Fibrinogen / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection; D014407:Tumor Cells, Cultured
D007976:Leupeptins; D046911:Macromolecular Substances; D013462:Sulfur Radioisotopes; D000643:Ammonium Chloride; D002738:Chloroquine; D005340:Fibrinogen; D008715:Methionine; C032854:leupeptin