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J. Biol. Chem., Vol. 277, Issue 39, 36363-36372, September 27, 2002
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From the Departments of The Limulus Factor C (FC), a
multidomain glycoprotein that binds bacterial endotoxin with high
affinity, belongs to the serine protease family of the complement and
blood coagulation cascade. Here, we provide compelling evidence for the
importance of modular arrangement and relevance of the proline-rich
region (PRR) and N-glycosylation to the secretion and
function of FC. We propose that PRR could be a universal conformational
domain that regulates protein folding and targeting. FCs lacking PRR
preceding the serine protease domain, were localized intracellularly.
Misfolded conformers of the intracellular FCs were more susceptible to
trypsin digestion. Glycosylation inhibition studies indicate that the
presence but not the exact structure of the N-glycans
affects the secretion of FC, although the complexity of glycosylation
may influence its endotoxin-induced proteolytic cleavage with resultant
enzymatic activity. Disruption of specific N-glycan sites
at positions 740, 767, and 912, downstream of the PRR, at or near the
serine protease domain, blocks its secretion. Co-expressed molecular
chaperones like canine calnexin associates with glycosylated FCs to
increase its solubility and secretion level but did not alter their
expression profiles. Our results clearly demonstrate that the folding
and secretion of a multidomain serine protease like FC are determined by its modular domain arrangement and site-specific
N-glycans. The secreted FCs containing the N-terminal
portion of FC are able to detect lipopolysaccharide with high
sensitivity. We also identified the lectin-like and sushi 4 domains to
contribute to the binding of lipopolysaccharide.
Modular Arrangement and Secretion of a Multidomain
Serine Protease
EVIDENCE FOR INVOLVEMENT OF PROLINE-RICH REGION AND
N-GLYCANS IN THE SECRETION PATHWAY*
§¶,
,
Biological Sciences and
** Microbiology, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
*
This work was supported by National Science and Technology
Board Grant NSTB/LS/99/004.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Institut de Biologie Animale, Bâtiment
de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland.
To whom correspondence should be addressed: National University
of Singapore, Marine Biotechnology Laboratory, Dept. of Biological Sciences, 14 Science Dr. 4, Singapore 117543, Singapore. Tel.: 65-8742776; Fax: 65-67792486; E-mail: dbsdjl@nus.edu.sg.
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