Effects of removing the negatively charged N-terminal region of the salivary acidic proline-rich proteins by human leucocyte elastase.

Human leucocyte elastase from inflammatory gingival crevicular exudates (gingival crevicular fluid) contacts saliva and saliva-coated tooth surfaces coronal to the gingival margin. Major components of saliva are the salivary acidic proline-rich proteins (PRPs). These acidic PRPs, via the numerous negatively charged amino acid residues located predominantly within their amino-terminal region, bind to the hydroxyapatite mineral of the tooth surface and become part of the salivary pellicle. Thus the potential for human leucocyte elastase-mediated removal of the negatively charged amino-terminal region of acidic PRP variants (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f) was examined. It was determined that each of the acidic PRP variants was susceptible to fragmentation by human leucocyte elastase, in which the 16 amino-terminal segment was removed, leaving the respective residual fragment named as the transitional product (tr). The transitional products were termed PRP-1tr, PRP-2tr (PIF-str), PRP-3tr and PRP-4tr (PIF-ftr). Each of the residual transitional products of acidic PRP had an amino-terminal beginning with serine residue no. 17, determined by amino acid sequencing. When samples of human leucocyte elastase-treated acidic PRPs were placed on native polyacrylamide gels and electrophoresed, the respective transitional products moved more slowly than the parental acidic PRP molecules, reflecting the loss of a portion of the negatively charged section. In comparison to the acidic PRPs, the acidic PRP transitional products had markedly reduced binding to hydroxyapatite. The transitional products were resistant to further enzymatic digestion as a function of increased incubation time and appeared to exert an antihuman leucocyte elastase effect. However, when increased concentrations of human leucocyte elastase were incubated with the acidic PRP, a more extensive digestion occurred, leaving a residual peptide with an amino-terminal beginning with alanine residue no. 44. Interestingly, intact acidic PRPs if prebound to hydroxyapatite particles, resisted digestion by human leucocyte elastase. In summary, human leucocyte elastase was capable of digesting fluid-phase (unbound) acidic PRP in a manner that eliminated part of their negatively charged region, which subsequently reduced their binding to hydroxyapatite. High concentrations of human leucocyte elastase, arriving from inflammatory gingival crevicular exudates, may interrupt the normal binding of fluid-phase acidic PRPs to hydroxyapatite.