Proteolytic processing of a human salivary proline-rich protein precursor by proprotein convertases

Salivary proline-rich proteins (PRPs) are synthesized as precursors that ar e cleaved before secretion giving rise to glycosylated PRPs which have lubr icating function and basic PRPs which are potent precipitators of dietary t annins. The putative cleavage sites in the precursors for basic and glycosy lated PRPs all conform to the sequence RSXR down arrowS (X can be A, S or P ) in agreement with the recognition sequence (RXXR down arrow) for various proprotein convertases. PRB4S, a proprotein giving rise to a basic PRP (IB- 5) as well as a glycosylated PRP (II-1) was synthesized by in vitro transcr iption-translation. It was cleaved by furin at RSAR down arrowS(173-178) gi ving rise to two proteins II-1 and IB-5. Similarly another precursor with t he sequence RSAR down arrowS(173-178) was also cleaved by furin. This toget her with previous results show that in vitro furin can cleave all RSXR down arrowS sequences in the proproteins that give rise to glycosylated and bas ic PRPs. To demonstrate cellular cleavage, a human submandibular cell line (HSG) was transfected with a vector encoding PRB4S. This resulted in secret ion of II-1 and IB-5. The degree of cleavage was enhanced by coexpressing f urin and PRB4S. No cleavage occurred if the cells expressed a mutant PRB4S, R177Q, where the furin cleavage site had been destroyed. Cleavage was also inhibited if a furin inhibitor was coexpressed with PRB4S. Incubating the cells at 20 degreesC which blocks exit of proteins from the trans-Golgi net work demonstrated that cleavage occurs before exit of the proteins from thi s network. These results show that furin may be responsible for in vivo cle avage of PRP precursors. Transfecting furin-deficient RPE.40 cells with a v ector encoding PRB4S also led to secretion of II-1 and IB-5 showing that co nvertases other than furin can also cleave PRB4S in tissue culture.