A synonymous mutation in LMAN1 creates an ectopic splice donor site and causes combined deficiency of FV and FVIII.
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ABSTRACT:
SUBMITTER: Zhu M
PROVIDER: S-EPMC3563766 | biostudies-literature | 2012 Nov
REPOSITORIES: biostudies-literature
Publications
A synonymous mutation in LMAN1 creates an ectopic splice donor site and causes combined deficiency of FV and FVIII.
Zhu M M DAS V V Zheng C C Majumdar S S Zhang B B
Journal of thrombosis and haemostasis : JTH 20121101 11
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Project description:The LMAN1-MCFD2 (lectin, mannose binding 1/multiple coagulation factor deficiency protein 2) cargo receptor complex transports coagulation factors V (FV) and VIII (FVIII) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). LMAN1 (ERGIC-53) is a hexameric transmembrane protein with a carbohydrate recognition domain (CRD) on the ER luminal side. Here, we show that mutations in the first beta sheet of the CRD abolish MCFD2 binding without affecting the mannose binding, suggesting that LMAN1 interacts with MCFD2 through its N-terminal beta sheet, consistent with recently reported crystal structures of the CRD-MCFD2 complex. Mutations in the Ca(2+)- and sugar-binding sites of the CRD disrupt FV and FVIII interactions, without affecting MCFD2 binding. This interaction is independent of MCFD2, as LMAN1 mutants defective in MCFD2 binding can still interact with FVIII. Thus, the CRD of LMAN1 contains distinct, separable binding sites for both its partner protein (MCFD2) and the cargo proteins (FV/FVIII). Monomeric LMAN1 mutants are defective in ER exit and unable to interact with MCFD2, suggesting that the oligomerization of LMAN1 is necessary for its cargo receptor function. These results point to a central role of LMAN1 in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII.
| S-EPMC3031414 | biostudies-literature
Project description:The type 1-transmembrane protein LMAN1 (ERGIC-53) forms a complex with the soluble protein MCFD2 and cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Mutations in either LMAN1 or MCFD2 cause the combined deficiency of factor V (FV) and factor VIII (FVIII; F5F8D), suggesting an ER-to-Golgi cargo receptor function for the LMAN1-MCFD2 complex. Here we report the analysis of LMAN1-deficient mice. Levels of plasma FV and FVIII, and platelet FV, are all reduced to ∼ 50% of wild-type in Lman1(-/-) mice, compared with the 5%-30% levels typically observed in human F5F8D patients. Despite previous reports identifying cathepsin C, cathepsin Z, and α1-antitrypsin as additional potential cargoes for LMAN1, no differences were observed between wild-type and Lman1(-/-) mice in the levels of cathepsin C and cathepsin Z in liver lysates or α1-antitrypsin levels in plasma. LMAN1 deficiency had no apparent effect on COPII-coated vesicle formation in an in vitro assay. However, the ER in Lman1(-/-) hepatocytes is slightly distended, with significant accumulation of α1-antitrypsin and GRP78. An unexpected, partially penetrant, perinatal lethality was observed for Lman1(-/-) mice, dependent on the specific inbred strain genetic background, suggesting a potential role for other, as yet unidentified LMAN1-dependent cargo proteins.
| S-EPMC3179404 | biostudies-literature
Project description:Mutations in lectin, mannose-binding 1 (LMAN1) and multiple coagulation factor deficiency protein 2 (MCFD2) cause the combined deficiency of factor V (FV) and FVIII (F5F8D). LMAN1 and MCFD2 form a protein complex that transports FV and FVIII from the endoplasmic reticulum (ER) to the Golgi. Although both proteins are required for the cargo receptor function, little is known about the specific roles of LMAN1 and MCFD2 in transporting FV/FVIII. We used different LMAN1 and MCFD2 deficient cell lines to investigate the LMAN1/MCFD2-dependent FV/FVIII secretion pathway. LMAN1 deficiency led to more profound decreases in FV/FVIII secretion in HEK293T and HepG2 cells than in HCT116 cells, suggesting that regulation of cargo transport by the LMAN1/MCFD2 pathway varies in different cell types. Using these cell lines, we developed functional assays to accurately assess the pathogenicity of recently reported potential LMAN1 and MCFD2 missense mutations. LMAN1 with mutations abolishing carbohydrate binding can still partially rescue FV/FVIII secretion, suggesting that N-glycan binding is not essential for FV/FVIII transport. Surprisingly, overexpression of either wild-type or mutant MCFD2 is sufficient to rescue FV/FVIII secretion defects in LMAN1 deficient cells. These results suggest that cargo binding and transport are carried out by MCFD2 and that LMAN1 primarily serves as a shuttling carrier of MCFD2. Finally, overexpression of both LMAN1 and MCFD2 does not further increase FV/FVIII secretion, suggesting that the amount of the LMAN1-MCFD2 receptor complex is not a rate-limiting factor in ER-Golgi transport of FV/FVIII. This study provides new insight into the molecular mechanism of F5F8D and the intracellular trafficking of FV and FVIII.
| S-EPMC10119632 | biostudies-literature
Project description: Not available
| S-EPMC5466846 | biostudies-literature
Project description:Combined deficiency of factor V (FV) and factor VIII (FVIII) (F5F8D) is a genetic disorder characterized by mild-to-moderate bleeding and coordinate reduction in plasma FV and FVIII levels, as well as platelet FV level. Recent studies identified mutations in two genes (LMAN1 and MCFD2) as the cause of F5F8D. Though clinically indistinguishable, MCFD2 mutations generally exhibit lower levels of FV and FVIII than LMAN1 mutations. LMAN1 is a mannose-specific lectin that cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment. MCFD2 is an EF-hand domain protein that forms a calcium-dependent heteromeric complex with LMAN1 in cells. Missense mutations in the EF-hand domains of MCFD2 abolish the interaction with LMAN1. The LMAN1-MCFD2 complex may serve as a cargo receptor for the ER-to-Golgi transport of FV and FVIII, and perhaps a number of other glycoproteins. The B domain of FVIII may be important in mediating its interaction with the LMAN1-MCFD2 complex.
| S-EPMC2777536 | biostudies-literature
Project description:LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca(2+)-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca(2+) concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca(2+) concentrations. Our results suggest that compartmental changes in Ca(2+) concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.
| S-EPMC3711315 | biostudies-literature
Project description:BackgroundThe coagulation factors (F)V and VIII are homologous proteins that support hemostasis through their regulation of FX activity. Hemophilia A (HA) patients have reduced FVIII activity and a prolonged bleeding time that is corrected through the administration of exogenous FVIII. Around one-third of severe HA patients develop FVIII neutralizing antibodies, known as "inhibitors," which neutralize FVIII activity and preclude them from further FVIII therapy.ObjectivesWe hypothesized that, based on the degree of homology between FV and FVIII (~40%), FVIII-neutralizing antibodies could cross react with FV. To test this hypothesis, a panel of recombinant, patient-derived, FVIII-neutralizing antibodies were screened for cross-reactivity against FV.MethodsFactor V and FVIII activity was measured using one-stage clotting assays; structural analysis was carried out using a structural approach.ResultsWe detected FV neutralizing activity with the anti-FVIII A2 domain antibody NB11B2. Because this antibody was derived from an HA inhibitor patient, FV-neutralizing activity was then evaluated in a number of HA inhibitor patient plasma samples; nine alloimmune samples had FV-neutralizing activity whereas no FV neutralizing activity was found in the two autoimmune samples available. We next examined the degree of surface homology between FV and FVIII and found that structural similarity could explain the cross reactivity of the anti-A2 antibody and likely accounts for the cross reactivity we observed in patient samples.ConclusionsAlthough this novel observation is of interest, further work will be needed to determine whether FV neutralization in HA patient samples contributes to their bleeding diathesis.
| S-EPMC8360109 | biostudies-literature
Project description:BACKGROUND: Mutations in MECP2 are the main cause of Rett Syndrome. To date, no pathogenic synonymous MECP2 mutation has yet been identified. Here, we investigated a de novo synonymous variant c.48C>T (p.Gly16Gly) identified in a girl presenting with a typical RTT phenotype. METHODS: In silico analyses to predict the effects of sequence variation on mRNA splicing were employed, followed by sequencing and quantification of lymphocyte mRNAs from the subject for splice variants MECP2_E1 and MECP2_E2. RESULTS: Analysis of mRNA confirmed predictions that this synonymous mutation activates a splice-donor site at an early position in exon 1, leading to a deletion (r.[=, 48_63del]), codon frameshift and premature stop codon (p.Glu17Lysfs*16) for MECP2_E1. For MECP2_E2, the same premature splice site is used, but as this is located in the 5'untranslated region, no effect on the amino acid sequence is predicted. Quantitative analysis that specifically measured this cryptic splice variant also revealed a significant decrease in the quantity of the correct MECP2_E1 transcript, which indicates that this is the etiologically significant mutation in this patient. CONCLUSION: These findings suggest that synonymous variants of MECP2 as well as other known disease genes-and de novo variants in particular- should be re-evaluated for potential effects on splicing.
| S-EPMC3729535 | biostudies-literature
Project description:What proportion of coding sequence nucleotides have roles in splicing, and how strong is the selection that maintains them? Despite a large body of research into exonic splice regulatory signals, these questions have not been answered. This is because, to our knowledge, previous investigations have not explicitly disentangled the frequency of splice regulatory elements from the strength of the evolutionary constraint under which they evolve. Current data are consistent both with a scenario of weak and diffuse constraint, enveloping large swaths of sequence, as well as with well-defined pockets of strong purifying selection. In the former case, natural selection on exonic splice enhancers (ESEs) might primarily act as a slight modifier of codon usage bias. In the latter, mutations that disrupt ESEs are likely to have large fitness and, potentially, clinical effects. To distinguish between these scenarios, we used several different methods to determine the distribution of selection coefficients for new mutations within ESEs. The analyses converged to suggest that ∼15%-20% of fourfold degenerate sites are part of functional ESEs. Most of these sites are under strong evolutionary constraint. Therefore, exonic splice regulation does not simply impose a weak bias that gently nudges coding sequence evolution in a particular direction. Rather, the selection to preserve these motifs is a strong force that severely constrains the evolution of a substantial proportion of coding nucleotides. Thus synonymous mutations that disrupt ESEs should be considered as a potentially common cause of single-locus genetic disorders.
| S-EPMC6169883 | biostudies-literature
Project description:Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mtb genes. We explored the role of codon pair bias by recoding Mtb genes ( rpoB, mmpL3, ndh ) and assessing their expression in the closely related and tractable model organism M. smegmatis . To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation, but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which in turn led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation.ImportanceMycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb . We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is the first report that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.
| S-EPMC10055193 | biostudies-literature