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%
(Rfree 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
Cα 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.