The Critical Interaction of Glycoprotein (GP) Ibβ With GPIX—A Genetic Cause of Bernard-Soulier Syndrome

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2968-2975 ◽  
Author(s):  
Dermot Kenny ◽  
Patricia A. Morateck ◽  
Joan C. Gill ◽  
Robert R. Montgomery
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary Cells ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Single Nucleotide ◽  
Platelet Surface ◽  
Single Nucleotide Deletion ◽  
Nucleotide Deletion ◽  
Bernard Soulier Syndrome

Bernard-Soulier syndrome is an uncommon bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP)Ib/IX complex. The complex is composed of four subunits, GPIb, GPIbβ, GPIX, and GPV. Here we describe the molecular basis of a novel Bernard-Soulier syndrome variant in a patient in whom GPIb and GPIX were undetectable on the platelet surface. DNA sequence analysis showed normal sequence for GPIb, GPIX, and GPV. The GPIbβ gene has been mapped to the 22q11.2 region of chromosome 22 which was deleted from one chromosome of this patient. There was a single nucleotide deletion within the codon for Ala 80 in GPIbβ within the other allele. This mutation causes a translational frame shift that encodes for 86 altered amino acids and predicts a premature stop 15 amino acids short of the length of the wild-type protein. Transient coexpression of the mutant GPIbβ in 293T cells with wild-type GPIb and GPIX resulted in the surface expression of GPIb, but the absence of GPIX. Moreover, when a plasmid encoding the wild-type GPIbβ was transiently transfected into Chinese hamster ovary cells stably expressing GP, which retain the capacity to reexpress GPIX, there was a significant increase in the surface expression of GPIX. In contrast, when the mutant GPIbβ was transiently transfected into these cells, GPIX was not reexpressed on the plasma surface. Thus, a deletion of one copy of GPIbβ and a single nucleotide deletion in the codon for Ala 80 within the remaining GPIbβ allele causes the Bernard-Soulier phenotype through an interaction of GPIbβ with GPIX resulting in the absence of GPIb on the plasma membrane. The interaction of GPIbβ with GPIX is essential for the functional expression of GPIb.

The Critical Interaction of Glycoprotein (GP) Ibβ With GPIX—A Genetic Cause of Bernard-Soulier Syndrome

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2968-2975 ◽  
Author(s):  
Dermot Kenny ◽  
Patricia A. Morateck ◽  
Joan C. Gill ◽  
Robert R. Montgomery
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary Cells ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Single Nucleotide ◽  
Platelet Surface ◽  
Single Nucleotide Deletion ◽  
Nucleotide Deletion ◽  
Bernard Soulier Syndrome

Abstract Bernard-Soulier syndrome is an uncommon bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP)Ib/IX complex. The complex is composed of four subunits, GPIb, GPIbβ, GPIX, and GPV. Here we describe the molecular basis of a novel Bernard-Soulier syndrome variant in a patient in whom GPIb and GPIX were undetectable on the platelet surface. DNA sequence analysis showed normal sequence for GPIb, GPIX, and GPV. The GPIbβ gene has been mapped to the 22q11.2 region of chromosome 22 which was deleted from one chromosome of this patient. There was a single nucleotide deletion within the codon for Ala 80 in GPIbβ within the other allele. This mutation causes a translational frame shift that encodes for 86 altered amino acids and predicts a premature stop 15 amino acids short of the length of the wild-type protein. Transient coexpression of the mutant GPIbβ in 293T cells with wild-type GPIb and GPIX resulted in the surface expression of GPIb, but the absence of GPIX. Moreover, when a plasmid encoding the wild-type GPIbβ was transiently transfected into Chinese hamster ovary cells stably expressing GP, which retain the capacity to reexpress GPIX, there was a significant increase in the surface expression of GPIX. In contrast, when the mutant GPIbβ was transiently transfected into these cells, GPIX was not reexpressed on the plasma surface. Thus, a deletion of one copy of GPIbβ and a single nucleotide deletion in the codon for Ala 80 within the remaining GPIbβ allele causes the Bernard-Soulier phenotype through an interaction of GPIbβ with GPIX resulting in the absence of GPIb on the plasma membrane. The interaction of GPIbβ with GPIX is essential for the functional expression of GPIb.

Differential Pathogenesis of Bernard-Soulier Syndrome-Associated Mutations In the Platelet Glycoprotein Ibβ Ectodomain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3202-3202
Author(s):  
Wenjun Yang ◽  
P.A McEwan ◽  
Xi Mo ◽  
Jonas Emsley ◽  
Renhao Li
Keyword(s):  
Cho Cells ◽  
Disulfide Bonds ◽  
Surface Expression ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Interfacial Region ◽  
Support Surface ◽  
Wild Type ◽  
Platelet Surface ◽  
Bernard Soulier Syndrome

Abstract Abstract 3202 Eight missense mutations in the ectodomain of glycoprotein (GP)Ibβ have been identified in patients with Bernard-Soulier syndrome (BSS) that is characterized by the deficiency of functional GPIb-IX complex on the platelet surface, clearly highlighting the importance of GPIbβ ectodomain in assembly of the GPIb-IX complex. To understand the molecular pathogenesis of these mutations, we have characterized their effects on the expression, secretion, folding of the isolated GPIbβ ectodomain as well as its interaction with GPIX ectodomain in the context of full-length complex. Each of the 8 mutations — C5Y, R17C, P29L, N64T, P74R, Y88C, P96S, and A108P — was constructed into genes encoding HA-tagged GPIbβ ectodomain or full-length GPIbβ subunit, and the mutant gene transfected transiently, along with GPIba and GPIX genes if desired, into Chinese hamster ovary (CHO) cells. Flow cytometry and Western blot analysis indicated that while all 8 mutations impeded formation of the disulfide bonds between GPIba and GPIbβ and significantly decreased the surface expression level of GPIb-IX complex comparing to the wild-type, the extent of disruption varies with each mutation. Further characterization in the context of isolated GPIbβ ectodomain revealed that the majority of 8 mutations — C5Y, R17C, P29L, N64T, Y88C, P96S — are detrimental to proper folding of the GPIbβ ectodomain, resulting in secretion defects and/or domain misfolding. In contrast, two mutations, P74R and A108P, preserved structural integrity of the GPIbβ ectodomain since the mutant ectodomains exhibited wild-type-like secretion levels and formed no inter-molecular disulfide bonds. However, neither of the two mutations, in the context of full-length GPIbβ, were able to support surface expression of GPIX in transfected CHO cells as the wild-type, indicating that P74R and A108P disrupt the interaction between GPIbβ and GPIX ectodomains. Thus, our results demonstrated although all 8 BSS mutations in GPIbβ share the same phenotype, they impair expression of the GPIb-IX complex by two different mechanisms — disrupting folding of the GPIbβ ectodomain or disrupting interactions between GPIb-IX subunits. Furthermore, our results suggest that Pro74 and Ala108 may be located in the interfacial region between GPIbβ and GPIX ectodomains, helping to shed light on the structure of GPIb-IX complex. Disclosures: No relevant conflicts of interest to declare.

The cysteine knot of platelet glycoprotein Ibβ (GPIbβ) is critical for the interaction of GPIbβ with GPIX

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4428-4433 ◽  
Author(s):  
Dermot Kenny ◽  
Patricia A. Morateck ◽  
Robert R. Montgomery
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Mammalian Cells ◽  
Surface Expression ◽  
Terminal Region ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
N Terminus ◽  
Bernard Soulier Syndrome

The glycoprotein Ib (GPIb) complex is composed of GPIbα covalently attached to GPIbβ and noncovalently complexed with GPIX and GPV. Patients with Bernard-Soulier syndrome demonstrate that mutations in either GPIbβ or GPIX result in an absence of platelet GPIbα. This occurs through the interaction of GPIX with GPIbβ. The precise sites of interaction of GPIbβ with GPIX are not known. To characterize the interaction of GPIbβ and GPIX, we developed an anti-GPIbβ monoclonal antibody MBC 257.4, whose epitope was in the N-terminal region of GPIbβ. N-terminal truncations of GPIbβ were expressed in mammalian cells. N-terminal truncations of GPIbβ, missing the first 14, 26, or 31 amino acids, were surface-expressed but did not enable coexpressed GPIX to be surface expressed, suggesting that the site of interaction with GPIX was modified by these deletions. GPIbβ and GPIX chimeras corresponding to predicted boundaries were used to define the sites of interaction of GPIbβ with GPIX. Replacing the N-terminal disulfide loops of GPIbβ (amino acids 1-14) with the corresponding disulfide loops of GPIX (amino acids 1-22) resulted in surface expression of coexpressed wildtype GPIX. However, when the N terminus of GPIbβ was replaced to residue 32 with the N terminus of GPIX (amino acids 1-36), GPIX did not surface express with this chimera. These results suggest that the cysteine knot region of GPIbβ in the N terminus is critical for the conformation of GPIbβ that interacts with GPIX and further suggests that a critical interaction of GPIbβ with GPIX involve residues 15 through 32 of GPIbβ.

The genetic defect in two well-studied cases of Bernard-Soulier syndrome: a point mutation in the fifth leucine-rich repeat of platelet glycoprotein Ib alpha

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3805-3814 ◽  
Author(s):  
C Li ◽  
SE Martin ◽  
GJ Roth
Keyword(s):  
Point Mutation ◽  
Genetic Defect ◽  
Pcr Amplification ◽  
Mutant Gene ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Molecular Defect ◽  
Wild Type ◽  
Giant Platelets ◽  
Bernard Soulier Syndrome

Bernard-Soulier syndrome (B-Ss) is a rare congenital bleeding disorder caused by abnormal giant platelets, thrombocytopenia, and defective glycoprotein (GP) Ib-V-IX, the adhesion receptor for von Willebrand factor (vWF). This report describes the molecular defect in two related individuals with well-established B-Ss whose platelets exhibit decreased GPIb-IX and normal GPV on their surfaces. The GPIb-V-IX genes of the two patients were analyzed by Southern blotting, hetero-duplex analysis, and polymerase chain reaction (PCR) amplification/sequencing. A point mutation was found in codon 129 of the GPIb alpha gene that results in the substitution of proline for leucine in the first position of the fifth leucine-rich glycoprotein repeat of the mature gene product. The mutation (CTC: leucine, wild-type to CCC: proline, mutant) eliminates a Sac I restriction site, facilitating analysis of the mutation in the propositi (both homozygotes), unaffected family members (8 heterozygotes and 8 wild-type), and 58 normal controls (116 wild-type alleles). The status of the genomes was confirmed by the sequencing of platelet cDNA. The mutation does not affect transcription of the Ib-IX genes, as estimated by PCR and Northern blot analysis, but it does inhibit surface expression of the receptor as assessed by transient transfection of mutant and wild-type GPIb alpha genes into mouse Ib beta-IX L cells. Many of the cells (43%) transfected with the normal gene express surface GPIb alpha, whereas untransfected cells and those transfected with the mutant gene lack surface GPIb alpha entirely. Patient platelets were tested both for vWF binding in the presence of ristocetin and for surface GPIb-IX expression. In these instances, despite their inability to agglutinate with ristocetin and vWF, patient platelets exhibit about 40% of normal vWF binding and 40% of normal Ib-IX surface antigens. The results suggest that the described mutation (GPIb alpha: Leu129 -> Pro) affects the conformation of the GPIb-V-IX receptor, alters its availability on platelet surfaces, and causes the observed Bernard-Soulier phenotype.

Quaternary Organization of GPIb-IX Complex and Insights Into Bernard-Soulier Syndrome Revealed by the Crystal Structures of GPIbβ Ectodomain and a GPIbβ/GPIX Chimera

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2195-2195
Author(s):  
Paul A. McEwan ◽  
Wenjun Yang ◽  
Katherine H. Carr ◽  
Xi Mo ◽  
Xiaofeng Zheng ◽  
...  
Keyword(s):  
Structural Model ◽  
Conflicts Of Interest ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Surface ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Ovary Cells ◽  
Bernard Soulier Syndrome

Abstract Abstract 2195 Platelet glycoprotein (GP)Ib-IX receptor complex contains three subunits, GPIbα, GPIbβ and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome (BSS). We have crystallized the GPIbβ ectodomain (GPIbβE) and determined the structure to reveal a single leucine-rich repeat with N- and C-terminal disulfide bonded capping regions. The central region of the structure can be divided into concave parallel β-sheet and convex loops. The crystal structure of a GPIbβE/GPIXE chimera that contains three non-continguous convex loops of GPIX and retains a GPIbβ-binding site of GPIX (Mo et al. J. Thromb. Haemost. 7:1533–40, 2009) was also determined. The chimera, but not GPIbβE, forms a homotetramer in the crystal, revealing a quaternary interface between GPIbβ and GPIX ectodomains. Central to this interface is residue Tyr106 from GPIbβ that inserts into a shallow and largely hydrophobic pocket generated by two convex loops from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIbβ and GPIX in the full-length complex. Eight GPIbβ missense mutations identified from BSS patients were examined in transiently transfected Chinese hamster ovary cells for changes to the GPIb-IX complex surface expression. Six of the eight mutations lead to secretion defect and/or misfolding of GPIbβE. In contrast, the other two mutations, A108P and P74R, were found to maintain normal secretion and folding of GPIbβE but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbβE interface with GPIX indicates that residues Ala108 and Pro74 in GPIbβ are located at the GPIbβE/GPIXE interfaces. Based on the tetrameric arrangement of the chimera structure, we propose a structural model for the GPIb-IX complex that embodies its organizing principles and helps to provide mechanistic insights on its assembly, function and regulation. Disclosures: No relevant conflicts of interest to declare.

A Novel Missense Mutation Asp69Gly in the Platelet Glycoprotein Ib(beta) Gene in Two Families with Bernard-Soulier Syndrome,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3300-3300
Author(s):  
Wolf A Hassenpflug ◽  
Belinda Weber ◽  
Tobias Obser ◽  
Ulrich Budde ◽  
Reinhard Schneppenheim
Keyword(s):  
Surface Expression ◽  
Cell Surface Expression ◽  
Platelet Glycoprotein ◽  
Facs Analysis ◽  
Platelet Surface ◽  
Minimal Trauma ◽  
Recurrent Epistaxis ◽  
The Impact ◽  
Beta Gene ◽  
Bernard Soulier Syndrome

Abstract Abstract 3300 Bernard-Soulier Syndrome (BSS) is characterized by macrothrombocytopenia and impaired platelet function due to defects of the GpIb-IX-V complex. Patients with Bernard-Soulier Syndrome are rare and only few disease causing mutations have been identified in the platelet gylcoprotein Ib(alpha), Ib(beta) or glycoprotein IX genes, respectively. We characterized 2 independent families with Bernard-Soulier Syndrome. Affected members of both families presented with a bleeding history including gross hematoma after minimal trauma, recurrent epistaxis, menorrhagia and prolonged bleeding after surgery. Routine laboratory tests showed reduced platelet numbers and increased platelet volume. Consistent with the diagnosis of Bernard-Soulier syndrome FACS analysis demonstrated reduced GpIb-IX expression on the platelet surface. Platelet agglutination in the presence of ristocetin (RIPA) was impaired. Using standard techniques we sequenced the GpIb(alpha), GpIb(beta) and GpIX genes. We identified a novel mutation Asp69Gly in the GpIb(beta) gene in both families. The mutation was homozygous in two children of the first family and one child of the second family. As expected, the parents of the affected index patients were heterozygous mutation carriers. To further investigate the impact of the Asp69Gly mutation on GpIb expression we used structure prediction software and homology-based protein modeling. Asp69Gly lies within an extracellular region flanking a leucin-rich element and cysteine-cluster. It affects the highly conserved center aspartic acid of a CDC motif that is thought to form one of two cystein-loops close to the transmembrane domain. We propose that this mutation disrupts the regular conformation of the cystein loop, potentially leading to abnormal protein folding and protein decay in the endoplasmic reticulum. In addition to a mere defect of biosynthesis our data hint at additional functional consequences of the Asp69Gly mutation. The father and the mother of the index patient in the first family, who are heterzygous for the Asp69Gly mutation, express only slightly reduced levels of platelet GpIb-IX based on FACS analysis. Nevertheless both of them suffer from epistaxis and menorrhagia, respectively. Expression studies are underway to clarify the influence of the Asp69Gly mutation on GpIb expression and function. Preliminary data show reduced cell surface expression of the GPIb-IX complex and reduced binding to von Willebrand-factor (VWF). While the functional and structural effects of mutations in the GpIb(alpha) and GpIX genes have been thoroughly studied, the impact of mutations in the GpIb(beta) gene is less clear. Our results could help to elucidate how the GpIb(beta) protein contributes to the assembly and surface expression of the GpIb-IX-V complex. Disclosures: No relevant conflicts of interest to declare.

Surface expression of glycoprotein Ibα is dependent on glycoprotein Ibβ: evidence from a novel mutation causing Bernard-Soulier syndrome

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 532-539 ◽  
Author(s):  
Niamh Moran ◽  
Patricia A. Morateck ◽  
Adele Deering ◽  
Michelle Ryan ◽  
Robert R. Montgomery ◽  
...  
Keyword(s):  
Novel Mutation ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Confocal Imaging ◽  
Stable Cell Line ◽  
Platelet Glycoprotein ◽  
Gene Encoding ◽  
Platelet Surface ◽  
Platelet Receptor ◽  
Bernard Soulier Syndrome

Abstract Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb, GPIbβ, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb was not present on the platelet surface, GPIb was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb. DNA-sequence analysis revealed normal sequences for GPIb and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIbβ, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIbβ, together with wild-type GPIb and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary  IX cells, a stable cell line expressing GPIb that retains the capacity to re-express GPIX. Thus, we found that GPIbβ affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb and GPIX into the platelet membrane.

Surface expression of glycoprotein Ibα is dependent on glycoprotein Ibβ: evidence from a novel mutation causing Bernard-Soulier syndrome

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 532-539 ◽  
Author(s):  
Niamh Moran ◽  
Patricia A. Morateck ◽  
Adele Deering ◽  
Michelle Ryan ◽  
Robert R. Montgomery ◽  
...  
Keyword(s):  
Novel Mutation ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Confocal Imaging ◽  
Stable Cell Line ◽  
Platelet Glycoprotein ◽  
Gene Encoding ◽  
Platelet Surface ◽  
Platelet Receptor ◽  
Bernard Soulier Syndrome

Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb, GPIbβ, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb was not present on the platelet surface, GPIb was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb. DNA-sequence analysis revealed normal sequences for GPIb and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIbβ, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIbβ, together with wild-type GPIb and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary  IX cells, a stable cell line expressing GPIb that retains the capacity to re-express GPIX. Thus, we found that GPIbβ affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb and GPIX into the platelet membrane.

scholarly journals Identification of a novel homozygous loss-of-function mutation in FUCA1 gene causing severe fucosidosis: A case report

2021 ◽  
Vol 49 (4) ◽  
pp. 030006052110059
Author(s):  
Xinwen Zhang ◽  
Shaozhi Zhao ◽  
Hongwei Liu ◽  
Xiaoyan Wang ◽  
Xiaolei Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lysosomal Storage Disorder ◽  
Autosomal Recessive ◽  
Signs And Symptoms ◽  
Lysosomal Storage ◽  
Loss Of Function ◽  
Wild Type ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Exon 1

Fucosidosis is a rare lysosomal storage disorder characterized by deficiency of α-L-fucosidase with an autosomal recessive mode of inheritance. Here, we describe a 4-year-old Chinese boy with signs and symptoms of fucosidosis but his parents were phenotypically normal. Whole exome sequencing (WES) identified a novel homozygous single nucleotide deletion (c.82delG) in the exon 1 of the FUCA1 gene. This mutation will lead to a frameshift which will result in the formation of a truncated FUCA1 protein (p.Val28Cysfs*105) of 132 amino acids approximately one-third the size of the wild type FUCA1 protein (466 amino acids). Both parents were carrying the mutation in a heterozygous state. This study expands the mutational spectrum of the FUCA1 gene associated with fucosidosis and emphasises the benefits of WES for accurate and timely clinical diagnosis of this rare disease.

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