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

Salivary proline-rich proteins (PRPs) are synthesized as precursors that are cleaved before secretion giving rise to glycosylated PRPs which have lubricating function and basic PRPs which are potent precipitators of dietary tannins. The putative cleavage sites in the precursors for basic and glycosylated PRPs all conform to the sequence RSXR↓S (X can be A, S or P) in agreement with the recognition sequence (RXXR↓) 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 transcription-translation. It was cleaved by furin at RSAR↓S(173–178) giving rise to two proteins II-1 and IB-5. Similarly another precursor with the sequence RSAR↓S(173–178) was also cleaved by furin. This together with previous results show that in vitro furin can cleave all RSXR↓S sequences in the proproteins that give rise to glycosylated and basic PRPs. To demonstrate cellular cleavage, a human submandibular cell line (HSG) was transfected with a vector encoding PRB4S. This resulted in secretion of II-1 and IB-5. The degree of cleavage was enhanced by coexpressing furin 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 °C which blocks exit of proteins from the trans-Golgi network demonstrated that cleavage occurs before exit of the proteins from this network. These results show that furin may be responsible for in vivo cleavage of PRP precursors. Transfecting furin-deficient RPE.40 cells with a vector encoding PRB4S also led to secretion of II-1 and IB-5 showing that convertases other than furin can also cleave PRB4S in tissue culture.