title: Analysis of fibrinogen gamma-chain truncations shows the C-terminus, particularly gammaIle387, is essential for assembly and secretion of this multichain protein.
abstract: To examine the role of the fibrinogen gamma chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated gamma chains, terminating between residues gamma379 and the C-terminus, gamma411. The variant fibrinogens were synthesized from altered gamma-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with gamma chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the gamma chain, from 1.4 microg/mL for normal fibrinogen to 0.39 microg/mL for gamma 387 fibrinogen. Immunoassays of cell lysates showed that all variant gamma chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated that the gamma-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the gamma chain are essential for fibrinogen assembly, and more specifically, that gamma387 is critical. We propose that the loss of residue gamma387 destabilized the structure of gamma chain, preventing assembly of alphagamma and betagamma dimers, essential intermediates in the assembly of normal fibrinogen.
authors: Laboratory of Clinical Chemistry, Department of Medical Technology, School of Allied Medical Sciences, Shinshu University, the Second Department of Internal Medicine, Shinshu University School of Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors: Nobuo
authors: N
authors: Okumura
authors: Fumiko
authors: F
authors: Terasawa
authors: Hitoshi
authors: H
authors: Tanaka
authors: Masako
authors: M
authors: Hirota
authors: Hiroyoshi
authors: H
authors: Ota
authors: Kiyoshi
authors: K
authors: Kitano
authors: Kendo
authors: K
authors: Kiyosawa
authors: Susan T
authors: ST
authors: Lord
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005340:Fibrinogen / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
chemical_list: D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007532:Isoleucine; D005340:Fibrinogen