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

Abstract

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 have been collected to 2.1 Å resolution, and the structure has been fully refined to an R-factor of 18.4%~R free 27.7%!. The ng-HBP structure reveals that neither the secondary nor tertiary structure have changed due to the removal of the glycosylation, as compared to the previously determined glycosylated HBP structure. Although the primary events in N-linked glycosylation occurs concomitant with polypeptide synthesis and therefore possesses the ability to influence early events in protein folding, we see no evidence of glycosylation influencing the structure of the protein. The root-mean-square deviation between the superimposed structures was 0.24 Å~on Ca atoms!, and only minor local structural differences are observed. Also, the overall stability of the protein seems to be unaffected by glycosylation, as judged by the B-factors derived from the two X-ray structures. The flexibility of a glycan site may be determined by the local polypeptide 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 peritonitis, glycosylated HBP treatment rescues mice from and an otherwise lethal injury. 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 to a lesser extent than glycosylated HBP.

Analysis of individual azurocidin N-glycosylation sites in regard to its secretion by insect cells, susceptibility to proteolysis and antibacterial activity.

“…3). It has been shown previously that a complete lack of glycosylation strongly reduced the biological activity of azurocidin, since the nonglycosylated protein mediated a limited stimulation of LPS-induced cytokine release from monocytes (Iversen et al, 1999). Although the lack of N-glycosylation at individual sites did not significantly influence the antibacterial activity, all these data support the hypothesis that the oligosaccharide moiety of human azurocidin may play a role in its biological function.…”

“…This effect could be explained by the function of Asn-linked oligosaccharides in the overall protein stability and/or folding. Iversen et al (1999) showed that the lack of glycosylation did not affect the folding of azurocidin, as the secondary and tertiary structures of non-glycosylated recombinant azurocidin and wild-type recombinant azurocidin were nearly identical. The surface loops to which some glycans are attached were also unaffected by the removal of the glycosylation.…”

Proteomic Analysis of Human Acute Leukemia Cells

“…It was negative in all cases of ALL. Azurocidin (17) and cationic antimicrobial peptides (ref. 18; Fig.…”

“…After matching the micropreparative gel image with the analytical image, in-gel digestion was performed with a previously published protocol (17).…”

A Promising Candidate: Heparin-Binding Protein Steps onto the Stage of Sepsis Prediction

“…The putative fourth carbohydrate attachment site is located in the region between residues 89 and 114 [5]. The biological activity of HBP in regard to mediating LPS-induced IL-6 release from monocytes is severely reduced by the removal of N-glycosylation [25]. Interestingly, however, the loss of HBP glycosylation does not affect the folding, secondary and tertiary structure, or stability of HBP [25].…”