Role of the Leucine-Rich Domain of Platelet GPIbα in Correct Post-translational Processing – The Nancy I Bernard-Soulier Mutation Expressed on CHO Cells

Summary

The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbα found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbα were transfected into CHO cells expressing GPIbβ and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPIbαΔLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient’s platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbαΔLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbα leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOαΔLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOαβIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOαΔLeuβIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbα is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbα in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.

Keywords

Glycoprotein - recombinant - glycosylation - von Willebrand factor - adhesion

• References

• 1 Kroll MH, Hellums JD, McIntire LV, Schaefer AI, Moake JL. Platelet and shear stress. Blood 1996; 88: 1525-41.
• 2 Ikeda Y, Handa M, Kamata T, Kawano K, Kawai Y, Watanabe K, Kawakami K, Sakai K, Fukuyama M, Itagaki I, Yoshioka A, Ruggeri ZM. Transmembrane calcium influx associated with von Willebrand factor binding to GPIb in the initiation of shear induced platelet aggregation. Thromb Haemost 1993; 69: 496-502.
• 3 Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84: 289-97.
• 4 Sadler JE, Matsushita T, Dong Z, Tuley EA, Westfield LA. Molecular mechanism and classification of von Willebrand disease. Thromb Haemost 1995; 74: 161-6.
• 5 George JN, Nurden AT. Inherited disorders of the platelet membrane: Glanzmann thrombasthenia, Bernard-Soulier syndrome and other disorders. Hemostasis and Thrombosis. Basic Principles and Clinical Practice. Colman RW, Hirsh J, Marder VJ, Salzman EW. eds. Philadelphia: J. B. Lippincott; 1994: 652-672.
• 6 de la Salle C, Lanza F, Cazenave J-P. Biochemical and molecular basis of Bernard-Soulier syndrome: a review. Nouv Rev Fr Hématol 1995; 37: 215-22.
• 7 López JA, Andrew RK, Afshar-Kharghan V, Berndt MC. Bernard-Soulier syndrome. Blood 1998; 91: 4397-418.
• 8 Modderman PW, Admiraal LG, Sonnenberg A, von dem Borne AEGKr. Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane. J Biol Chem 1992; 267: 364-9.
• 9 López JA. The platelet glycoprotein Ib-IX complex. Blood Coag Fibrinolysis 1994; 05: 97-119.
• 10 López JA, Chung DW, Fujikawa K, Hagen FS, Papayanopoulou T, Roth GJ. Cloning of the α chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich α₂- glycoprotein. Proc Natl Acad Sci USA 1987; 84: 5615-9.
• 11 Wenger RH, Kieffer N, Wicki AN, Clemetson KJ. Structure of the human blood platelet membrane glycoprotein Ibα gene. Biochem Biophys Res Commun 1988; 156: 389-94.
• 12 López JA, Chung DW, Fujikawa K, Hagen FS, Davie EW, Roth GJ. The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence. Proc Natl Acad Sci USA 1988; 85: 2135-9.
• 13 Hickey MJ, Roth GJ. Characterization of the gene encoding human platelet glycoprotein IX. J Biol Chem 1993; 268: 3438-43.
• 14 Lanza F, Morales M, de la Salle C, Cazenave J-P, Clemetson KJ, Shimomura T, Phillips DR. Cloning and characterization of the gene encoding the human platelet glycoprotein V, a member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation. J Biol Chem 1993; 268: 20801-7.
• 15 Kobe B, Deisenhofer J. The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 1994; 19: 415-21.
• 16 Titani T, Takio K, Handa M, Ruggeri ZM. Amino acid sequence of the von Willebrand factor binding domain of platelet membrane glycoprotein Ib. Proc Natl Acad Sci USA 1987; 84: 5610-4.
• 17 De Marco L, Mazzucato M, Fabris F, de Roia D, Coser P, Girolami A, Vicente V, Ruggeri M Z. Variant Bernard-Soulier syndrome type Bolzano. A congenital bleeding disorder due to a structural and functional abnormality of the platelet glycoprotein Ib/IX complex. J Clin Invest 1990; 86: 25-31.
• 18 Miller JL, Lyle VA, Cunningham D. Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ibα leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease. Blood 1992; 79: 439-46.
• 19 Ware J, Russel S, Marchese P, Murata M, Mazzucato M, De Marco L, Ruggeri ZM. Point mutation in a leucine-rich repeat of platelet glycoprotein Ibα resulting in the Bernard-Soulier syndrome. J Clin Invest 1993; 02: 1213-20.
• 20 Furihata K, Yamamoto N, Yan J, Hasegawa Y, Nagasawa T, Tanoue K. An amino acid substitution of Phe for Leu¹²⁶ in a leucine rich repeat of GPIbα associated with a variant form of Bernard-Soulier syndrome. Blood 1994; 84: 476a (abstr).
• 21 Li C, Martin E, Roth GJ. The genetic defect in two well studied cases of Bernard-Soulier syndrome: a point mutation in the fifth leucine-rich repeat of platelet glycoprotein Ibα. Blood 1995; 86: 3805-14.
• 22 de la Salle C, Baas M-J, Lanza F, Schwartz A, Hanau D, Chevalier J, Gachet C, Briquel ME, Cazenave J-P. A three-base deletion removing a leucine residue in a leucine-rich repeat of platelet glycoprotein Ibα associated with a variant of Bernard-Soulier syndrome (Nancy I). Br J Haematol 1995; 89: 386-96.
• 23 de la Salle C, Lanza F, Caen J, Nurden AT, Juhan I, Briquel ME, Ternisien C, Conard J, Cazenave J-P. Molecular characterization of five different mutations in patients with Bernard-Soulier bleeding disorder. Central role of GPIbβ and GPIX in GPIb-IX-V expression. Thromb Haemost 1997; 77: 68 (abstr).
• 24 Wright SD, Michaelides K, Johnson DJD, West NC, Tuddenham EGD. Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier syndrome. Blood 1993; 81: 2339-47.
• 25 Clemetson JM, Kyrle PA, Brenner B, Clemetson KJ. Variant Bernard-Soulier syndrome associated with a homozygous mutation in the leucinerich domain of glycoprotein IX. Blood 1994; 84: 1124-31.
• 26 Noris P, Simsek S, Stibbe J, von dem Borne AEGKr. A phenylalanine-55 to serine amino-acid substitution in the human glycoprotein IX leucine-rich repeat is associated with Bernard-Soulier syndrome. Br J Haematol 1997; 97: 312-20.
• 27 Cazenave J-P, Hemmendinger S, Beretz A, Sutter-Bay A, Launay J. L’agrégation plaquettaire: outil d’investigation clinique et d’étude pharmacologique. Méthodologie. Annales de Biologie Clinique 1983; 41: 167-79.
• 28 Kinlough-Rathbone RL, Mustard JF, Packham MA, Perry DW, Reimers HJ, Cazenave J-P. Properties of washed human platelets. Thromb Haemost 1977; 37: 291-308.
• 29 Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site specific mutagenesis without phenotypic selection. Methods Enzymol 1987; 154: 367-82.
• 30 López JA, Leung B, Reynolds CC, Li CQ, Fox JEB. Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits. J Biol Chem 1992; 267: 12851-9.
• 31 Toti F, Gachet C, Ohlmann P, Stierlé A, Grunebaum L, Wiesel M-L, Cazenave J-P. Electrophoretic studies on molecular defects of von Willebrand factor and platelet glycoprotein IIb-IIIa with antibodies produced in egg yolk from laying hens. Haemostasis 1992; 22: 32-40.
• 32 Montgomery RR, Zimmermann TS. Von Willebrand’s disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand disease. J Clin Invest 1978; 61: 1498-507.
• 33 Montgomery RR, Kunicki TJ, Taves C, Pidard D, Corcoran M. Diagnosis of Bernard-Soulier syndrome and Glanzmann’s thrombasthenia with a monoclonal assay on whole blood. J Clin Invest 1993; 71: 385-9.
• 34 López J, Li CQ, Weisman SM, Chambers M. The glycoprotein Ib-IX complex-specific monoclonal antibody SZ1 binds to a conformationsensitive epitope on glycoprotein IX: implications for the target antigen of quinine/quinidine-dependent autoantibodies. Blood 1995; 85: 1254-8.
• 35 Ruan C, Du X, Xi X, Castaldi PA, Berndt MC. A murine antiglycoprotein Ib complex monoclonal antibody, SZ2, inhibits platelet aggregation induced by both ristocetin and collagen. Blood 1987; 69: 570-7.
• 36 McMichael AJ, Rust NA, Pilch JR, Sochinsky R, Morton J, Mason J, Ruan C, Tobelem G, Caen J. Monoclonal antibody to human platelet glycoprotein Ib. Br J Haematol 1981; 49: 501-9.
• 37 Handa M, Titani K, Holland LZ, Roberts JR, Ruggeri ZM. The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib: characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. J Biol Chem 1986; 261: 12579-85.
• 38 Murata M, Ware J, Ruggeri ZM. Site directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ibα demonstrating negatively charged residues involved in von Willebrand factor binding. J Biol Chem 1991; 266: 15474-80.
• 39 Berndt MC, Du X, Booth WJ. Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex. Biochemistry 1988; 27: 633-40.
• 40 Berndt MC, Gregory CB, Chong BH, Zola H, Castaldi PA. Molecular characterization of quinine/quinidine drug-dependent antibody platelet interaction using monoclonal antibodies. Blood 1983; 66: 1292-301.
• 41 Cooke BM, Usami S, Perry I, Nash GB. A simplified method for culture of endothelial cells and analysis of adhesion of blood cells under conditions of flow. Microvasc Res 1993; 45: 33-45.
• 42 Cranmer SL, Ulsemer P, Cooke BM, Salem HH, de la Salle C, Lanza F, Jackson SP. Glycoprotein (GP) Ib-IX transfected cells roll on a von Willebrand factor matrix under flow. J Biol Chem 1999; 274: 6097-106.
• 43 Kieffer N, Debili N, Wicki A, Titeux M, Henri A, Mishal Z, Breton-Gorius J, Vainchenker W, Clemetson KJ. Expression of platelet glycoprotein Ibα in HEL cells. J Biol Chem 1986; 261: 15854-62.
• 44 Remaley AT, Ugorski M, Wu N, Litzky L, Burger SR, Moore JS, Fukuda M, Spitalnik SL. Expression of human glycophorin A in wild type and glycosylation deficient Chinese Hamster Ovary cells. J Biol Chem 1991; 266: 24176-83.
• 45 Dong JF, Li CQ, López JA. Tyrosine sulfatation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding. Biochemistry 1994; 33: 13946-53.
• 46 Kajava AJ. Structural diversity of leucine-rich repeat proteins. J Mol Biol 1998; 277: 519-27.
• 47 Kobe B, Deisenhofer J. Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 1993; 366: 751-6.
• 48 Gralnick HR, Williams S, McKeown LP, Hansmann K, Fenton II JW, Krutzsch H. High affinity α-thrombin binding to platelet glycoprotein Ibα: identification of two binding domains. Proc Natl Acad Sci USA 1994; 91: 6334-8.
• 49 Dong JF, Hyun W, López JA. Aggregation of mammalian cells expressing the platelet glycoprotein Ib-IX complex and the requirement for tyrosine sulfation of GPIbα. Blood 1995; 86: 4175-83.
• 50 Moshfegh K, Lengweiler S, Häner M, Aebi U, Steiner B, Beer JH. Fine structural and functional consequences of deglycosylation of the platelet adhesion receptor GPIb-IX. Biochem Biophys Res Commun 1998; 249: 903-9.