Factor XDebrecen: Gly204Arg mutation in factor X causes the synthesis of a non-secretable protein and severe factor X deficiency

Due to a homozygous Gly204Arg mutation in the factor X (FX)gene no detectable FX antigen was found in the plasma of a one-yearold patient with severe bleeding diathesis. The amino acid replacementdestabilized the disulfide bond that holds the two FX chainstogether, decreasing the interaction between the Cys201-Cys206loop region and the region connecting the EGF2 and serine proteasedomains. Both Gly204 FX and Arg204 FX were synthesized in transfectedcells, but only the wild type protein became secreted. The mutantprotein was diverted from the normal secretory pathway and retainedat the trans Golgi-late endosome level.

Key words: factor X, factor X deficiency, inherited bleeding diathesis, rare coagulopathy, F10 mutation.

Introduction

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Introduction

Blood coagulation factor X (FX) is synthesized in the liveras a single chain protein.^1,2 During maturation, a tripeptide,Arg140-Lys141-Arg142, is cut out from the peptide chain in theGolgi. The resultant 17-kDa light-chain and 45-kDa heavy-chainare held together by a disulfide-bridge between Cys132 and Cys302.The light chain contains a Gla domain, two epidermal growthfactor (EGF) domains and an additional connecting region (CR).The heavy chain consists of the serine protease (SP) domainand the N-terminal activation peptide (AP) that is cleaved offupon activation by factors IXa or VIIa. Inherited FX deficiencyis a rare (1:1,000,000) coagulopathy with severe bleeding symptomsin homozygous patients.¹ More than 80 mutations have been describedin the F10 gene (www.med.unc.edu/isth/mutations-databases),the majority of which are missense mutations. In type I FX deficiency,both functional activity and antigen level are decreased, whilein type II deficiency a dysfunctional molecule is present. Afew reports have suggested that secretion defects, due to missensemutations in domains of the mature protein, cause type I deficiency.However, this has been proved by expression studies.^3–5Here we demonstrated that Gly204Arg substitution resulted insevere type I FX deficiency by causing structural changes inthe molecule and a secretion defect due to retention at thetrans Golgi-late endosome level.

Design and Methods

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Design and Methods

Patient
A one-year-old boy with subdural hematoma and bruising had highlyprolonged prothrombin time and activated partial thromboplastintime. The activity of all clotting factors, except for FX, wasin the reference interval. A highly reduced FX activity (1%)together with FX antigen concentration below the limit of detectionsuggested type I FX deficiency. No bleeding history in the familywas reported. The study protocol was approved by the EthicsCommittee of the University. Informed consent was given by thepatient’s mother.

Procedures
With the exception of exon 8, primers described in the literaturewere used for amplification of genomic DNA.^3,6,7 The followingprimers were designed for the amplification of exon 8: 5'-CGTCTGTCCCAGGGGAC-3'(forward), 5'-TGGGATCTCACTTTAATGGAG-3' (reverse). Forward andreverse direct fluorescent sequencing of PCR products were performedby ABI PRISM 310 DNA sequencer (Perkin-Elmer, Foster City, CA,USA). The mutant FX expression vector⁸ was generated by site-directedmutagenesis of the wild type FX cDNA inserted in pCMV4 vectorusing QuickChange site-directed mutagenesis kit (Stratagene,La Jolla, CA, USA). Human embryonic kidney (HEK293) cells weretransiently transfected⁸ with 1 Ìg wild type or mutantvector using Effectene transfection reagent (Qiagen). Aftera 72-hour incubation conditioned media were collected, partof the cells were used for confocal laser scanning microscopy(CLSM), while another part of the cells was lysed in a buffercontaining 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NonidetP40, 0.5% sodium deoxycholate and a protease inhibitor cocktail(Roche). Aliquots of the media and cell lysates were used forFX antigen determination by ELISA (Asserachrom X:Ag, DiagnosticaStago, Asnières, France) and for immunoprecipitationby biotinylated goat anti-human FX antibody (Haematologic TechnologiesInc, Vermont, VT) and strep-tavidine agarose (Sigma-Aldrich).Immunoprecipitates were analyzed by Western blotting using rabbitanti-human FX antibody (Dako, Glostrup, Denmark) and VectastainElite ABC kit (Vector Laboratories, Burlingame, CA, USA). Thereaction was visualized by ECL Plus chemiluminescence reagent(Amersham, Little Chalfont, UK).

For pulse chase analysis, transfected HEK 293 cells were preincubatedwith methionine-, and FCS-free medium for 30 min. Then theywere pulse-labeled for 30 min with 0.5 mCi/mL [³⁵S]methionine(Amersham), and chased for 30 min, 2 and 6 hr. FX immunoprecipitatedfrom cell lysates and culture media was analyzed by SDS PAGEand fluorography.

Cells fixed in 96% ethanol, 1% acetic acid were incubated with5% normal human serum in phosphate buffered saline for 15 minto prevent non-specific IgG binding. Staining for FX was combinedwith the detection of calnexin (an endoplasmic reticulum marker),or mannosidase II (a cis-Golgi marker), or mannose 6 phosphatereceptor (a trans-Golgi-late endosome marker).^9–11 Cellpreparations were examined by CLSM (LSM 410, Zeiss, Oberkochen,Germany). The relative amount of FX was determined by measuringintegrated fluorescence intensity on 150–200 cells/slide.¹²Molecular modeling and simulations were performed on reconstitutedsingle-chain (des1–45)FX.^13,14 After minimization andheating up dynamics, the geometries of Gly204 and Arg204 EGF2-CR-AP-SPconstruct were submitted to molecular dynamics simulation usingOPLS/AA force field and periodic boundary conditions with TIP4Pwater solvent model with the GROMACS package.¹⁵

Results and Discussion

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Results and Discussion

Molecular genetic analysis revealed a homozygous G>A missensemutation at coding nucleotide position c.730 resulting in aGly204Arg replacement in the amino acid sequence. The motherwas heterozygous for the mutation; the father was unknown andother family members were not available for testing. The absenceof the mutation in one hundred healthy individuals suggestedits causative nature. We have given a preliminary presentationof this mutation¹⁶ and later it was also detected in an Iranianpatient.⁵

Using ELISA technique and quantitative fluorescent image analysis,in HEK cells expressing Arg204 FX the concentration of FX wasfound to be 1.5-fold and 2.2-fold higher respectively than incells expressing wild type FX. In agreement with these results,the band representing Arg204 FX on the Western blot was moreintense than the band corresponding to Gly204 FX (Figure 1A).FX appeared as a double band which is probably due to the differentextent of glycosylation.^2,17 In the media of cells expressingwild type FX 93.7 mU/mL FX was measured by ELISA, while FX couldnot be detected in the media of cells expressing the mutantprotein. Immediately after pulse labeling of HEK cells, theband of wild type FX was more intense than the band representingArg204 mutant (Figure 1B). During the chase period the amountof radio-labeled wild type FX rapidly decreased in the celllysate, and, in parallel, bands representing FX heavy and lightchains appeared in the culture media. In contrast, the intensityof the band corresponding to the mutant protein increased duringthe first 30 min in the cell lysate and only then started todecay. No mutant FX appeared in the culture media. Evidently,newly synthesized wild type FX became secreted and rapidly disappearedfrom the cells, while the mutant proteins, which cannot be secreted,accumulated in the cytoplasm and were finally eliminated byintracellular proteases. Double immunofluorescent staining showedthat the localization of Arg204 FX in HEK cells neither correspondedto the localization of the endoplasmic reticulum (Figures 2Ba–c)nor to that of the cis-Golgi apparatus (Figures 2Bd–f).However, the staining for Arg204 FX appeared in co-localizationwith the trans-Golgi-late endosome network (Figures 2Bg–i).No such co-localization was found in the case of wild type protein(Figures 2Ag–i). The results suggest that the mutant FXis diverted from the normal secretory pathway toward the lateendosomes, and finally becomes degraded in lysosomes.

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Figure 1. Detection of factor X expressed by HEK293 cells transfected with wild type and mutant factor X constructs. A. Detection of FX in the cell lysate (1–3) and culture media (4–6) by Western blotting. SDS PAGE was performed in non-reducing condition. 1,4: non-transfected cells; 2,5: cells expressing wild type FX; 3,6: cells expressing Arg204 mutant FX; 7: purified FX standard. FX in the cell lysate had slightly higher mobility than FX in the culture media or FX prepared from human plasma, the difference probably reflects different stages of glycosylation. B. Metabolic labeling of HEK cells expressing wild type and mutant factor X. Non-transfected (NT) HEK293 cells and cells transfected with wild type (W) and Arg204 mutant (M) constructs were pulse labeled with [³⁵S] methionine, then chased for various intervals. Cell lysate samples represent immunoprecipitates from ~5x10⁵ cells, while culture media samples were immunoprecipitated from the supernatant of ~2.5x10⁵ cells. The immunoprecipitates loaded onto the gels derived from the same amount of proteins in each sample. HC: FX heavy chain; LC: FX light chain.

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Figure 2. Double immunofluorescent staining for FX and intracellular organelle markers in HEK cells expressing wild type (A) or Arg204 mutant (B) factor X. Goat anti-human factor X antibody (Haematologic Technologies), followed by biotinylated anti-goat IgG (Vector) and Texas Red Streptavidin (Vector) were used to detect FX. Monoclonal primary antibodies against calnexin and mannose 6 phosphate receptor and rabbit antibody against mannosidase II were from Abcam (Cambridge, UK). The respective secondary antibodies (Vector) were labeled with fluorescein. Confocal laser scanning microscopic images (magnification 100 x 3,2). a–c: immunostaning for FX ${alpha}$ and the endoplasmic reticulum marker, calnexin; d–f: immunostaning for FX and mannosidase II, a cis-Golgi marker; g–i: immunostaning for FX and mannose-6P receptor, a trans-Golgi marker. FX (a,d,g) appears in red; calnexin (b), mannosidase II (e) and mannose 6P receptor (h) appear in green; c,f and i are merged images.

The site of the mutation is located in a small disulphide-bridgedloop formed between Cys201 and Cys206. Cys201, Gly204 and Cys206residues were highly conserved during evolution and they arealso present in factor VII, the closest relative of FX (SwissProtdatabase). From in silico experiments (Figure 3), it is evidentthat the replacement of small apolar glycine at position 204by the bulky positively charged arginine introduces major structuralchanges into its surroundings. The region between Cys132 andArg142, which corresponds with the C-terminal part of the lightchain plus the tripeptide, has different orientation and liesmuch farther from the Cys201-Cys206 disulphide bridged loopthan in wild type FX. The mean Coulomb interaction energy betweenthe loop and the Cys132-Arg142 peptide segment was lower inthe wild type protein than in the Arg204 mutant (–188.6kcal/mol versus –88.5 kcal/mol), i.e. in the case of themutant the interaction considerably weakened. The Cys132 residueis involved in the formation of the disulfide bond that keepsthe EGF2 and SP domains together, the mutation could preventthe formation of this disulfide bond or make it unstable andmight in this way also influence the global folding of the protein.The structural changes could also alter the molecular recognitionand/or transport properties of FX and contribute to the secretiondefect of Arg204 mutant.

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Figure 3. Averaged three-dimensional structure for wild type (A) and Gly204Arg mutant (B) factor X. The starting structure obtained from the Research Collaboratory for Structural Bioinformatics, Protein Data Bank (www.rcsb.org) was supplemented according to Venkateswarlu et al.¹⁴ The geometries of Gly204 and Arg204 EGF2-CR-AP-SP constructs were submitted to a 7 nanosecond molecular dynamics simulation (6 nanoseconds equilibration and 1 nanosecond sampling). Results were visualized by the Visual Molecular Dinamics software suit.¹⁸ Parts of epidermal growth factor 2 (transparent green), activation peptide (steal blue), serine protease (transparent pink) domains and the whole connecting region (gold) are shown on the figure. The Arg140-Lys141-Arg142 tripeptide is depicted in yellow. Selected amino acid residues as well as the disulfide bonds Cys132-Cys302 and Cys201-Cys206 are shown by ball-and-stick model. The location of Cys201-Cys206 loop is shown in opaque pink. The distance between the 204 C and 137 C atoms is shown by a double-headed red arrow.

Acknowledgments

wild type FX cDNA inserted in pCMV4 vector was a generous giftfrom Prof. Katherine A. High and Dr. Rodney M. Camire (Children’sHospital of Philadelphia, PA, USA)

Footnotes

Authorship and Disclosures

ZB was involved in all aspects of research, data analysis andwriting the paper; HB and RÁ performed and evaluatedimmunofluorescent studies; IK performed molecular modeling;CK and ÉA were involved in the laboratory diagnosis andmanagement of the patient; GH was involved in immunoblottingexperiments; LM designed research, analyzed the data and wrotethe paper.

The authors reported no potential conflicts of interest.

Funding: this study was supported by grants from the HungarianNational Research Fund (OTKA-NKTH NI 69238 and OTKA K62087),from the Hungarian Academy of Sciences (MTA 2006TKI227) andfrom the Hungarian Ministry of Health (ETT 406/2006).

Received for publication May 19, 2007. Accepted for publication November 28, 2007.

References

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