Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX.
Biochem J
; 358(Pt 2): 295-303, 2001 Sep 01.
Article
in En
| MEDLINE
| ID: mdl-11513727
ABSTRACT
The multisubunit leucine-rich glycoprotein (GP) Ib-IX-V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard-Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse-chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbalpha undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbalpha and surface expression of the complex. Biosynthetic studies were then performed on a Bernard-Soulier variant based on previous description of abnormal GPIbalpha size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbalpha. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments ('ERGIC') and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Protein Processing, Post-Translational
/
Platelet Glycoprotein GPIb-IX Complex
Limits:
Animals
Language:
En
Journal:
Biochem J
Year:
2001
Document type:
Article
Affiliation country: