THE MYELOID DIFFERENTIATION ANTIGEN CD14 IS N-GLYCOSYLATED AND O-GLYCOSYLATED - CONTRIBUTION OF N-LINKED GLYCOSYLATION TO DIFFERENT SOLUBLECD14 ISOFORMS

The myeloid differentiation antigen CD14 acts as the major receptor fo r bacterial lipopolysaccharide (LPS). A soluble form of the protein (s CD14) is present in human serum which functions as a soluble LPS recep tor. We have compared the isoform patterns of soluble CD14 derived fro m human serum and of the recombinant proteins produced by CHO cells tr ansfected with either the wild-type CD14 gene or with a cDNA coding fo r a truncated protein which lacks the C-terminal 21 amino acids [sCD14 -(1-335)-peptide]. Using SDS/PAGE, two dominant isoforms (53 and 50 kD a) and two minor forms (46 and 43 kDa) can be detected in serum as wel l as in the supernatants of both transfectants. sCD14 is a glycoprotei n which carries N- and O-linked carbohydrates. The different isoforms of sCD14-(1-335)-peptide are due to differences in the content of N-li nked sugars. However after the removal of N- and O-linked carbohydrate s from serum- and CHO-derived wild-type proteins, two isoforms are sti ll present. These results indicate that N-linked glycosylation contrib utes to but does not fully explain the different forms of soluble CD14 . We further examined whether the mutation of individual N-linked glyc osylation sites influences the expression of membrane-bound and solubl e CD14 forms and the ability of the membrane-bound molecule to bind LP S. As with the wild-type proteins, the different isoforms of the solub le mutants are partially due to differences in N-linked glycosylation. A truncated mutant which lacks the two N-terminal glycosylation sites {[Asp18, Asp132]CD14-(1-335)peptide} does not give rise to multiple f orms on SDS gels. Like CD14-(1-335)-peptide, this mutant is not expres sed on the cell surface suggesting that a smaller isoform present in t he wild-type preparations results from proteolytic cleavage of the mem brane-bound molecule. N-linked carbohydrates do not seem to be importa nt for the binding of LPS to membrane-bound CD14.