Analysis of fibrinogen γ-chain truncations shows the C-terminus, particularly γIle387, is essential for assembly and secretion of this multichain protein

Abstract

To examine the role of the fibrinogen ␥ chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated ␥ chains, terminating between residues ␥379 and the C-terminus, ␥411. The variant fibrinogens were synthesized from altered ␥-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with ␥ chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the ␥ chain, from 1.4 g/mL for normal fibrinogen to 0.39 g/mL for ␥ 387 fibrinogen. Immunoassays of cell lysates showed that all variant ␥ 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 ␥-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 ␥ chain are essential for fibrinogen assembly, and more specifically, that ␥387 is critical. We propose that the loss of residue ␥387 destabilized the structure of ␥ chain, preventing assembly of ␣␥ and ␤␥ dimers, essential intermediates in the assembly of normal fibrinogen. IntroductionFibrinogen is a 340-kd plasma glycoprotein consisting of 2 copies of 3 polypeptide chains, A␣, B␤, and ␥, linked by an extensive network of 29 intrachain and interchain disulfide bonds. 1,2 A separate gene encodes each polypeptide chain. The 3 chains are synthesized, assembled into the 6-chain molecule, and secreted from hepatocytes into the plasma. Studies of fibrinogen expressed from the endogenous genes in human hepatocytes or from transfected complementary DNAs (cDNAs) in baby hamster kidney (BHK) cells have shown that assembly occurs through specific intermediates. 3,4 These intermediates, ␣␥ dimers, ␤␥ dimers, and ␣␤␥ half-molecules, all include the ␥ chain.Normal fibrinogen levels are 2 to 4 mg/mL in plasma. Hypofibrinogenemia or afibrinogenemia, defined as reduced or immeasurable levels of fibrinogen in plasma, can be hereditary. Although the first case of afibrinogenemia was reported in 1920, 5 the genetic basis of these abnormalities was first demonstrated in 1999. 6 Since that time, 26 different mutations have been identified in cases of afibrinogenemia or hypofibrinogenemia. [7][8][9][10][11][12][13][14][15][16][17][18][19] These mutations were found in all 3 genes, and include missense, nonsense, and frameshift mutations; splice-site abnormalities; and large deletions. Thus, fibrinogen deficiencies are associated with a variety of genetic chan...

Nonsense-mediated mRNA decay was demonstrated in two hypofibrinogenemias caused by heterozygous nonsense mutations of FGG, Shizuoka III and Kanazawa II

“…In the absence of CHX, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of CHX, levels of mRNAs transcribed from the mutant gene increased dose-dependently. 3 Finally, these results demonstrated that aberrant mRNAs containing γ23X or γ376X are degraded by the NMD system and translation decrease in hepatocytes, resulting in hypofibrinogenemias.…”

“…We have already reported that the length of the γ-chain should be at least 387 residues for fibrinogen assembly and secretion 3) . To confirm that lower plasma fibrinogen levels of Shizuoka III and Kanazawa II than normal had been caused by shorter γ-chains, we prepared vectors containing γ23X-and γ376X-cDNAs and transfected them into CHO cells, which synthesize normal human fibrinogen Aα-and Bβ-chains.…”

“…SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions (10 % gel) or non-reducing conditions (8 % gel), and immunoblot analysis of fibrinogen or individual polypeptides were performed using enhanced chemiluminescence (ECL) detection reagents, and Hyperfilm-ECL (both from Amersham Pharmacia Biotech, Buckinghamshire, UK) as described previously [3].…”

“…We have already reported that C-terminal residues of the γ-chain have an important role in fibrinogen assembly and secretion; in particular, the required length of the γ-chain is 387 residues, and 387Ile is essential [3]. Thus, it was supposed that truncated mutations of γ23X and γ376X do not assemble into an intact fibrinogen molecule and are not secreted into the bloodstream.…”

Differences in the function and secretion of congenital aberrant fibrinogenemia between heterozygous γD320G (Okayama II) and γΔN319-ΔD320 (Otsu I)

“…Cells were cultured and colonies were selected on histidinol (Aldrich Chem. Co. Milwaukee, WI, USA), as described elsewhere [22]. Fibrinogen concentrations in cell lysates or culture media from the selected clones were measured by the enzyme-linked immunosorbent assay (ELISA), and the assembly of fibrinogen and/or synthesis of three polypeptide chains in the cell lysate was analyzed by SDS-PAGE and immunoblotting as described previously [22].…”