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

Abstract

SummaryThe 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.

Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases

“…GPIba is also a receptor for thrombin. The integrity of the LRR domain of GPIb is essential for normal processing and function of the GPIb-IX complex [196]. BSS is an autosomal recessive bleeding disorder caused by genetic abnormalities in the GPIb-V-IX complex [195].…”

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX

“…Mab WM23 against GPIbα was obtained from Dr Michael Berndt (Baker Medical Research Institute, Prahan, Victoria, Australia). Mab GS28 against GS28 (a specific cis-Golgi marker) was purchased from StressGen Biotechnologies Corp. (Victoria, BC, Canada), Mab CTR 433 (against an uncharacterized marker of the medial Golgi) and Mab Gi93 [against ER-to-Golgi intermediate compartments (ERGIC) marker] were obtained from Dr Michel Bornens (Institut Curie, Paris, France), and a polyclonal antibody against calnexin (an ER marker) was purchased from Chemicon, Temecula, CA, U.S.A. CHO cell lines expressing wild-type GPIbα associated with GPIbβ and GPIX (CHOαβIX) or expressing Leu"(*-deleted GPIbα associated with GPIbβ and GPIX (CHOα∆LeuβIX) have been described previously [20].…”

“…GPV was omitted on the basis of previous reports that it is not required for efficient expression of the complex [12,13]. The biosynthetic study was extended to a Bernard-Soulier mutant receptor characterized by a major defect in oligosaccharide processing and transport to the cell surface [20], despite normal synthesis and assembly of its three subunits. Results indicate that tight control is exerted at the exit to the endoplasmic reticulum (ER) and throughout passage through the Golgi structures, thus limiting the expression of abnormal complexes harbouring even subtle mutations on a single subunit.…”

Platelet Receptors and Their Role in Diseases

“…Those described so far were all mutations of GPIbα: L57F (10), A156V (16), and L179 deletions (17). Those described so far were all mutations of GPIbα: L57F (10), A156V (16), and L179 deletions (17).…”