As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 252 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. serum, which is directly secreted or derived from protease-dependent shedding of the membrane-bound molecule [l 1 -141. As a soluble LPS-receptor, sCD14 competes with mCDt4 for LPS binding and is able to neutralize LPS-induced responses in vitro and in vivo [7,[15][16][17]. In addition to this, however, sCD14 mediates the LPS-induced activation of non-CD14-expressing endothelial, epithelial and smooth-muscle cells 118-221.Because the LPS molecule and its receptor and carrier proteins are primary targets for therapeutic-intervention strategies, the knowledge of ligand-receptor interactions on a molecular level may provide a rationale for the development of specific drugs that interfere with LPS binding or with LPS signaling. Therefore, different approaches have been used to identify the LPS-binding domain of human CDI 4. Viriyakosol and Kirkland 1231 constructed a series of small deletion mutants of the hydrophilic regions within the first 65 residues of the mature protein. They could not demonstrate serum-dependent binding of 'H-labeled LPS to any of their mutant proteins, though only CHO cells transfected with a deletion mutant spanning amino acids 35-39 failed to respond to LPS by translocation of NFKB. By sequential deletion of the C-terminal part of the molecule, Juan et al. [24] showed that CD14-(1-152)-peptide is able to recognize LPS and to mediate cellular responses induced by LPS. In a further study from the same group, an epitope of CD14 was defined, which is protected by LPS from cleavage by endoproteinase Asp-N 1251. They showed that a deletion mutant covering amino acids 57-64 does not interact with LPS In this study we have performed an alanine scan of amino acids 1 to 152 of human CD14. 23 alanine-substitution mutants have been constructed and stably expressed in CHO cells. We analyzed whether mutant proteins expressed on the surface of 1261.

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.

ADP-ribosyltransferase activity was shown to be present on the surface of human monocytes. Incubating the cells in the presence of BSA leads to an increase in enzyme activity. The acceptor amino acid mainly responsible for the ADP-ribose bond was identified as a cysteine residue. An increase in ADP-ribosyltransferase activity was observed when cells were treated for 16 h with bacterial lipopolysaccharide (LPS). Possible candidates for catalysing the reaction are mono-ADP-ribosyltransferases (ARTs). When measuring expression of the mRNA of ART1, 3, 4 and 5, only ART3 mRNA was detected in unstimulated monocytes. Upon stimulation for 16 h with LPS, lipoteichoic acid or peptidoglycan, ART4 mRNA was found to be expressed. No ART4 signal appeared after a 4 h exposure of the cells to LPS. Cell-surface proteins were labelled when incubating monocytes with [(32)P]NAD(+). Their molecular masses were 29, 33, 43, 45, 60 and 82 kDa. In response to LPS an additional protein of 31 kDa was found to be labelled. The bound label was resistant to treatment with NH(2)OH but sensitive to HgCl(2), characteristic of a cysteine-linked ADP-ribosylation.

In recent years, evidence has accumulated that NAD+ serves as a precursor of metabolites that are involved in a number of regulatory processes. In this work we show that extracellularly added NAD+ was rapidly degraded by intact human monocytes to nicotinamide and ADP-ribose. Besides these main products, minor amounts of AMP, ADP and cADP-ribose were formed. Expression of CD38, which has been identified as NAD+-glycohydrolase (EC 3.2.2.6) degrading NAD+ into nicotinamide and ADP-ribose, was determined on freshly isolated human monocytes by flow cytometry and RT-PCR. Upon ligation with anti-CD38 mAb, CD38 underwent internalization, shedding and new expression. As monocytes possess an intracellular CD38 pool, it could serve as a source for newly expressed CD38. Differentiation of monocytes to macrophages resulted in down-regulation of surface expression of CD38. This decrease correlates with a reduction in NADase activity, indicating that the amount of functional active CD38 molecules decrease during differentiation. As CD38 mRNA was found to be diminished in macrophages, regulation of the gene product seems to occur at the level of transcription or mRNA stability.

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