Molecular cloning, expression and assembly of multimeric von Willebrand factor.
Recently, substantial progress has been made in our knowledge of the domains involved in correlating structure and function of vWF, as well as in the biosynthesis and assembly of multimeric vWF. These studies were greatly supported by the development of three new techniques. (1) In vitro culturing of (human) vascular endothelial cells has allowed studies on the subcellular localization for dimerization of pro-vWF subunits, multimerization, carbohydrate and proteolytic processing. Moreover, this approach has provided insight into the complex intracellular routing of vWF that proceeds by either one of two pathways. During the constitutive pathway, vWF is packaged in secretory vesicles that are rapidly secreted both at the luminal and at the basolateral side. The regulated pathway includes storage of vWF molecules in specialized organelles, i.e. the Weibel-Palade bodies. (2) The application of proteases to dissect purified multimeric vWF and to assign function(s) to defined proteolytic fragments of vWF. Monoclonal antibodies, raised against native vWF, that block specific functions and bind to fragments are subsequently employed to correlate structure and function. More precise localizations of functions are now feasible, using overlapping synthetic peptides derived from the primary amino acid sequence of (pro)-vWF and antibodies raised against such peptides. This approach has permitted a fine mapping of the interaction site of vWF with the platelet receptor glycoprotein (GP) IIB/IIIA. (3) Molecular cloning of full-length vWF cDNA and the development of eukaryotic expression systems have substantially increased the possibilities to investigate structures on pro-vWF involved in the biosynthesis and the assembly of multimers. In particular, the construction of point and deletion mutants of vWF, employing vWF cDNA, and subsequent expression in heterologous cells have demonstrated that proteolytic processing of pro-vWF, between arginine (Arg-763) and serine (Ser-764), to generate free propolypeptide and mature vWF is not required for multimerization. Finally, the propolypeptide has an obligatory role for the formation of multimers, enabling interdimer disulphide bonding of free sulphhydryl groups located within the mature vWF part of pro-vWF.
Pannekoek
H
H
Voorberg
J
J
D001704:Biopolymers; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006801:Humans; D013329:Structure-Activity Relationship; D014162:Transfection / Q000235:genetics; D014841:von Willebrand Factor / Q000096:biosynthesis / Q000235:genetics*
D001704:Biopolymers; D014841:von Willebrand Factor; D004247:DNA