Structure and function of the N-linked glycans of HBP/CAP37/azurocidin: Crystal structure determination and biological characterization of nonglycosylated HBP

The three N-glycosylation sites of human heparin binding protein (HBP) have been mutated to produce a nonglycosylated HBP (ng-HBP) mutant. ng-HBP has been crystallized and tested for biological activity. Complete X-ray data h ave been collected to 2.1 Angstrom resolution, and the structure has been f ully refined to an R-factor of 18.4% (R-free 27.7%). The ng-HBP structure r eveals that neither the secondary nor tertiary structure have changed due t o the removal of the glycosylation, as compared to the previously determine d glycosylated HBP structure. Although the primary events in N-linked glyco sylation occurs concomitant with polypeptide synthesis and therefore posses ses the ability to influence early events in protein folding, we see no evi dence of glycosylation influencing the structure of the protein. The root-m ean-square deviation between the superimposed structures was 0.24 Angstrom (on C alpha atoms), and only minor local structural differences are observe d. Also, the overall stability of the protein seems to be unaffected by gly cosylation, as judged by the B-factors derived from the two X-ray structure s. The flexibility of a glycan site may be determined by the local polypept ide sequence and structure rather than the glycan itself. The biological in vitro activity assay data show that ng-HBP, contrary to glycosylated HBP, mediates only a very limited stimulation of the lipopolysaccharide induced cytokine release from human monocytes. In animal models of fecal peritoniti s, glycosylated HBP treatment rescues mice from and an otherwise lethal inj ury. It appears that ng-HBP have significant effect on survival, and it can be concluded that ng-HBP can stimulate the host defence machinery albeit t o a lesser extent than glycosylated HBP.