Mutation of the –RPVR– Propeptide Motif at C-Terminal of the Aα Chain Is Associated with Decreased Fibrinogen Expression

Fibrinogen is synthesized from individual Aα, Bβ and γ chains that are directed into the endoplasmic reticulum of the hepatocyte by co-translationally cleaved signal peptides.[1] Dimeric (Aα-Bβ-γ)₂ molecules are assembled and transit to the Golgi where a propeptide is cleaved from the C-terminal (–⁶⁰⁴HAKSRPV R ^↓GIHTSPLGKPSLSP⁶²⁵) of the Aα chain.[2] Furin-mediated cleavage occurs at a canonical –4R–1R paired arginine motif[3] [4] and is followed by carboxypeptidase catalysed hydrolysis of the newly exposed R⁶¹¹ residue, to yield the mature chain terminating at V⁶¹⁰, while in the Golgi the Aα chain undergoes further phosphorylation and O-glycosylation before final secretion of the mature 340-kDa molecule into the circulation where its αC appendage forms a natively unfolded structure.[5]

In the case of proalbumin, failure of cleavage of the N-terminal propeptide through mutation of the dibasic motif does not affect export of the protein from the hepatocyte,[6] and failure of cleavage of another liver-derived protein, profactor IX (through mutation of either –1R > S or –4R > Q), results in haemophilia because the uncleaved precursor is unable to be activated by factor Xa.[7] [8] Similarly, mutation of the dibasic motif of the insulin proreceptor results in diabetes. Interestingly, the creation of an additional competent –4R–1R dibasic site at the N-terminal of fibrinogen Canterbury (Aα20Asp→Val) results in dysfibrinogenaemia due to excision of the A peptide and the GPR– ‘knob’ as the assembled protein transits the Golgi.[8] It is not however known if the normal fibrinogen propeptide has a specific function, or whether cleavage is necessary for efficient export from the hepatocyte.

Here we report a novel Aα608Arg→Leu substitution that prevents propeptide processing, is associated with decreased expression of molecules containing the affected chain and results in hypofibrinogenaemia. We also consider if the mutation might influence the balance between Aα and alternatively spliced αE chains, which usually make up less than 5% of the total.

The 17-year-old proband in this case had a history of low fibrinogen levels (∼1.5 g/L) and presented recently with recurrent epistaxis. Current evaluation showed a Clauss fibrinogen of 1.5 g/L, a physical level of 1.8 g/L (normal range: 1.5–4.0) and a thrombin clotting time (TCT) of 19.4 seconds (normal range: 15–21). Plasma proteins showed a normal pattern on agarose gel electrophoresis, but there was a decreased intensity of the fibrinogen band consistent with hypofibrinogenaemia. Further examination of purified fibrinogen on reducing SDS-PAGE showed a normal pattern of Aα, Bβ and γ chains[8] ([Fig. 1A]).

Fig. 1 (A) SDS PAGE of purified fibrinogen. Lanes 1, 2 and 4 controls, lane 3 proband; 7.5% reducing gel. (B) Deconvoluted electrospray TOF (time of flight) spectra of fibrinogen Aα chains. (a) Normal control with an unmodified Aα chain mass of 66,135 Da and higher masses reflecting phosphorylation (+80 Da) and O-glycosylation (+657 Da); (B) control heterozygous for Aα312Thr→Ala polymorphism (–30 Da); (c) proband heterozygous for a polymorphic Aα312Ala chain (66,105 Da) and for an extended Aα312Thr chain of 67,620 Da. (C) Schematic showing the C-terminal region of circulating Aα chains, the extended pro Aα chain and the αE chains. The predicted and measured masses of different genetic isoforms are shown. The Aα608Arg→Leu mutation in the Aa312Thr chain prevents propeptide cleavage leading to the production of 67,620 species.

Purified fibrinogen was further examined by online reversed-phase time-of-flight mass spectrometry (TOF MS).[9] [10] The proband had normal Bβ and γ chain spectra with masses of 54,213 and 48,383 Da exactly matching the predicted values of their monosialo isoforms (not shown). Extraction and deconvolution of Aα chain spectra showed one control had a mass of 66,135 Da, which was consistent with homozygosity for the common Aα312Thr allele (predicted translation mass of 66,132 Da); ([Fig. 1B](a)). The second control was heterozygous for the Aα312Thr→Ala polymorphism as reflected by the –30 Da stutter superimposed on the phosphorylation (+80 Da) and O-glycation (+657 Da) isoform pattern ([Fig. 1B](b)). With a signal at 66,105 Da, spectra from the proband suggested he was heterozygous for the Aα312Ala allele ([Fig. 1B](c)). However, repetition of the entire phosphorylation and O-glycation isoform pattern at +1515 Da suggested he had some form of protein extension encoded on his other allele. Also, the low ratio (0.16:1) of variant (67,620Da) to normal (66,105 Da) Aα chains implicates the mutation as the underlying cause of the hypofibrinogenaemia.

Direct amplification and deoxyribonucleic acid (DNA) sequencing of exon 5 of FGA confirmed the proband was heterozygous for the Aα312Thr→Ala polymorphism and indicated that he was also heterozygous for a novel NM_021871.2:c.1880G > T transversion (designated fibrinogen Murdoch). This predicts a 608Arg(CGC)→Leu(CTC) substitution just two residues in from the C-terminal (Val⁶¹⁰) of the mature circulating Aα chain and four residues in from the intron 5/6 alternative splice site.

These DNA sequence changes explain the presence of a normal Aα312Ala chain at 66,105 Da and showed the 608Arg→Leu mutation was present on the patient's 312Thr allele, and because the 608Arg→Leu substitution ablates the –4R–1R dibasic motif, the resultant protein should have an additional C-terminal extension of –RGIHTSPLGKPSLSP and a predicted overall mass of 67,618 Da ([Fig. 1C]). This was very close to the observed mass of 67,620 Da, and the absence of any Aα chains at 66,089 Da confirmed that no residual propeptide cleavage was occurring at the now monobasic Arg⁶¹⁰ site.

The low abundance of new extended Aα chain could be partially explained if some of the allele output was redirected into increased production of αE chains. The mutation itself is only 12 nucleotides upstream of the splice site that determines the relative proportion of Aα and αE chains produced, so we considered its possible effects on mRNA (messenger ribonucleic acid) editing. The c.1880G > T transversion is predicted to cause loss of an pre-mRNA-splicing factor 2/alternative splicing factor 1 (SF2/ASF) and acquisition of an SRp40 exonic splice enhancer (ESE) site (Alamut V2.7 Interactive-Biosoftware, Rouen, France). Wild-type score for SF2 binding is 2.3, but at 1.69 the variant score falls below the nominal 1.9 binding threshold, suggesting αE production would actually diminish. Offsetting this, the increased SRp40 score of 3.87 (threshold 2.68) predicts a more clearly defined GT splice site with increased exon 6 incorporation and αE production. We could, however, find no evidence of increased levels of αE chain translation, either by extraction of the ion chromatogram for a target mass of 92,878 Da (or 95,084 Da for the N-glycosylated chain) or by examination of reducing SDS-PAGE gels ([Fig. 1A]).

Together this suggests that protein from the variant allele is expressed at only 16% of the level of the normal allele and that cleavage of the propeptide might be important for efficient export of fibrinogen from the hepatocyte. The –4R–1R cleavage motif is well conserved within mammalian fibrinogen even to the extent of also having an additional –6K/R basic residue, which has been shown to increase the efficiency of furin-mediated cleavage of other proprotein targets.[4]

Western blots of baby hamster kidney (BHK) cells expressing Aα, Bβ and γ chains have shown that disruption of the dibasic site through mutation of 611Arg > Gly still allowed secretion of assembled molecules with the 15-residue propeptide still attached.[2] However, it was not possible from these experiments to assess if the actual amount of fibrinogen secreted was diminished or not. Further time course experiments in these BHK cells showed the rate of secretion of fibrinogen directly truncated after AαVal610 was the same as wild-type fibrinogen, suggesting the presence of the propeptide was not necessary for either assembly or secretion from these surrogate cells.[2] The conflicting implications of the BHK expression and the current in situ findings do raise the possibility that profibrinogen may be assembled and secreted more or less normally and that the proteins plasma turnover rate might be increased.

Further evidence from natural variants does, however, confirm the C-terminal sequence of the Aα chain is important for attaining normal plasma fibrinogen levels.[11] The FGA c.1846 delA (p.Thr616His fsX33) frame shift causes replacement of the C-terminal sequence from Thr⁵⁹⁷, with a novel -HIAPREAMLNLALSEVSTLLLWGSLPCPPRLS⁶⁴⁸ sequence containing a scrambled propeptide lacking a dibasic motif.[12] Heterozygotes from this Syrian family had fibrinogen concentrations of approximately 1 g/L, while homozygotes suffered from afibrinogenaemia. Similarly, in fibrinogen Grand Lyon (p.Tyr579Phe fsX99), where the last 66 amino acids are replaced by 72 new residues, the heterozygous frame shift causes hypofibrinogenaemia with antigenic levels of fibrinogen of only 1.06 g/L.[13]

These, and other Aα truncations with more profound sequence changes, have a clear impact on fibrinogen concentration. But what is clear from the findings here is that the simple point substitution (Aα608Arg→Leu) prevents propeptide cleavage, and is associated with both decreased allelic expression and hypofibrinogenaemia.

• References

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