The Myeloid Differentiation Antigen CD14 is N‐ and O‐Glycosylated

Abstract

The myeloid differentiation antigen CD14 acts as the major receptor for bacterial lipopolysaccharide (LPS). A soluble form of the protein (sCD14) is present in human serum which functions as a soluble LPS receptor. We have compared the isoform patterns of soluble CD14 derived from human serum and of the recombinant proteins produced by CHO cells transfected with either the wild-type CD14 gene or with a cDNA coding for a truncated protein which lacks the C-terminal 21 amino acids [sCD14-(1-335)-peptide]. Using SDSRAGE, two dominant isoforms (53 and 50 kDa) and two minor forms (46 and 43 kDa) can be detected in serum as well as in the supernatants of both transfectants. sCD14 is a glycoprotein which carries N-and 0-linked carbohydrates. The different isoforms of sCD14-(1-335)-peptide are due to differences in the content of N-linked sugars. However after the removal of N-and 0-linked carbohydrates from serum-and CHO-derived wild-type proteins, two isoforms are still present. These results indicate that N-linked glycosylation contributes to but does not fully explain the different forms of soluble CD14. We further examined whether the mutation of individual N-linked glycosylation sites influences the expression of membrane-bound and soluble CD14 forms and the ability of the membranebound molecule to bind LPS. As with the wild-type proteins, the different isoforms of the soluble mutants are partially due to differences in N-linked glycosylation. A truncated mutant which lacks the two Nterminal glycosylation sites { [Asplg, Asp132]CD14-(1-335)peptide) does not give rise to multiple forms on SDS gels. Like CD14-(I -335)-peptide, this mutant is not expressed on the cell surface suggesting that a smaller isoform present in the wild-type preparations results from proteolytic cleavage of the membrane-bound molecule. N-linked carbohydrates do not seem to be important for the binding of LPS to membrane-bound CD14.

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

“…1), indicating that the protein is expressed as a single isoform and in an endotoxin-free form, which is quite valuable for high-resolution NMR structural studies. The observed molecular weight is higher than the expected molecular weight of ∼18 kDa, which may be due to the presence of glycosylation at two potential N-glycosylation sites on sCD14-EBD, corresponding to Asn18 and Asn132 [21]. Deglycosylation studies with an enzyme PNGase F that deglycosylates N-glycosylated sites resulted in disappearance of the 32 kDa band and appearance of a single band at ∼20 kDa (data not shown), supporting the presence of glycosylation.…”

The production and characterization of anti-bovine CD14 monoclonal antibodies

“…These same cells also produce a high molecular mass CD14 receptor lacking the GPI anchor, which is directly exocytosed. Varying degrees of glycosylation also contribute to the heterogeneity of the sCD14 molecule [32].…”

“…recognizes and binds LPS has been determined [31,32,36], and most likely Gramnegative bacteria bind via LPS at their surface to the same site of CD14. Intact Gramnegative bacteria can bind to m-and sCD14 in the presence of serum [33], which indicates that LPS incorporated into the membrane of Gram negative bacteria can interact with CD14.…”

N-Linked Glycosylation at Asn3and the Positively Charged Residues within the Amino-Terminal Domain of the C1 Inhibitor Are Required for Interaction of the C1 Inhibitor withSalmonella entericaSerovar Typhimurium Lipopolysaccharide and Lipid A

“…Glycosylation has been shown to influence the activity of some endotoxin binding proteins, particularly MD-2 and TLR4 (9,37). Although CD14 is glycosylated, carbohydrate does not play a role in its function (52). Glycosylation of TLR4 at Asn 526 and Asn 575 is required for proper expression of the protein at the cell surface and therefore for appropriate function of the LPS receptor (9).…”