Characterization of Siglec-5 (CD170) expression and functional activity of anti-Siglec-5 antibodies on human phagocytes.

OBJECTIVE: The Siglec family of proteins consists of at least 10 members with immunoglobulin and lectin domains and with similar sialic acid-binding properties. Many Siglec family members are expressed on hematopoietic cells and are involved in cell/cell interactions. Some family members are suspected of regulating cellular processes through specific signaling pathways. Monoclonal antibodies were generated against specific epitopes of Siglec-5 (CD170) and were used to determine expression of Siglec-5 on normal blood and marrow cells and cell lines. The antibodies also were used to elucidate functional activity for Siglec-5 on blood neutrophils. METHODS: Flow cytometry and ELISA were used to determine the specificity of the monoclonal antibodies for Siglec-5 and to determine expression patterns. Chemiluminescence assays were employed to measure the oxidative burst activity of whole blood or purified neutrophils following treatment with the anti-Siglec-5 antibodies. RESULTS: Cell surface expression analysis demonstrated that the protein was expressed on gated human neutrophil and monocyte populations, both in the blood and bone marrow. Expression on neutrophils was enhanced by one-hour treatment with fMLP or TNF-alpha. Epitope-specific anti-Siglec-5 monoclonal antibodies did not directly activate human neutrophils; however, antibody binding augmented neutrophil oxidative burst activity as determined by fMLP-induced luminol-dependent chemiluminescence. CONCLUSION: Data demonstrating expression of Siglec-5 on cells of the myelomonocytic lineage and alteration of its expression by inflammatory stimuli suggest a role for this protein in cell/cell interactions following microbial exposure.

Structure of the C-terminally truncated human ProMMP9, a gelatin-binding matrix metalloproteinase.

The X-ray crystal structure of the proform of human matrix metalloproteinase MMP9 has been solved to 2.5 A resolution. The construct includes the prodomain, the catalytic domain and three FnII (fibronectin type II) domains. The prodomain is inserted into the active-site cleft, blocking access to the catalytic zinc. Comparison with the crystal structure of the most closely related MMP, MMP2, indicates that the conformations of residues in the active-site cleft and in the cysteine-switch peptide of the prodomain are highly conserved and that design of MMP9-specific inhibitors will be challenging. In common with MMP2, the MMP9 S1' inhibitor-binding pocket is large compared with that of other MMPs. One small point of difference in the S1' binding pockets of MMP9 and MMP2 may provide an opportunity to explore the design of specific inhibitors. The side chain of Arg424 in MMP9 is angled slightly away from the S1' pocket when compared with the corresponding residue in MMP2, Thr424. The secondary structure of the FnII domains is conserved between the two closely related MMPs, although the second FnII domain makes no contact with the catalytic domain in MMP9, while the same domain in MMP2 has a substantial area of interaction with the catalytic domain.