Structural Requirements for Catalysis, Expression, and Dimerization in the CD26/DPIV Gene Family

Abstract

Dipeptidyl peptidase IV (DP-IV/CD26), fibroblast activation protein (FAP), DP-like 1 (DPL1), DP8, DP9, and DPL2 comprise the CD26 gene family. CD26/DP-IV has roles in liver disease, T cell costimulation, chemokine biology, type II diabetes, and tumor biology. DPIV substrates include the glucagonlike peptides, neuropeptide Y, and the chemokines CCL3, CCL5, CCL11, CCL22, and CXCL12. We have proposed that the extracellular region of CD26 is analogous to prolyl oligopeptidase in consisting of an alpha/beta hydrolase domain contributed by both N- and C-terminal portions of the polypeptide and a seven-blade beta-propeller domain. Replacing the C-terminal portion of the predicted alpha/beta hydrolase domain of CD26 (residues 501-766) with the homologous portion of DP8 or DP9 produced intact proteins. However, these chimeric proteins lacked dimerization and peptidase activity, suggesting that CD26 dimerization requires the C-terminal portion of the alpha/beta hydrolase domain. Deleting some N-terminal residues of the alpha/beta hydrolase domain of CD26 ablated peptidase activity and greatly diminished cell surface expression. Together with previous data that CD26 peptidase activity requires the C-terminal 20 residues, this suggests that peptidase activity requires the entire alpha/beta hydrolase domain. The catalytic triad of DP8 was shown to be Ser(739)-Asp (817)-His(849). Glu(259) of DP8, a residue distant from the catalytic triad yet greatly conserved in the CD26 gene family, was shown to be required for peptidase activity. These data concord with our predicted CD26 structure, indicate that biosynthesis of a functional fragment of CD26 is difficult, and confirm the functional homology of DP8 with CD26.

Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line

“…Immunofluorescence (antibodies in Table 1) and second harmonic generation (SHG) on cryosections of human liver cirrhosis explants 17,18 and enzyme histochemistry, flow cytometry, and immunocytochemistry 19,20 have been described. Liver explants were obtained with informed consent and Royal Prince Alfred Hospital ethics committee approval.…”

“…Plasmid DNA extraction from Escherichia coli DH5␣ cells, site-directed mutagenesis, and human embryonic kidney (HEK) 293T cell line (ATCC, CRL-11268) transfection and analysis by enzyme stain and flow cytometry have been described previously. 19,20 293T cells were assayed 40 to 48 hours after transfection. The LX-2 cell line was maintained as described.…”

A Novel Role of Dipeptidyl Peptidase 9 in Epidermal Growth Factor Signaling

“…DPP9 was detected as a strong 80 kDa and a weaker 130 kDa band, whereas DPP8 was seen at approximately 110 and 150 kDa with similar band intensities. The DPP4 dimer runs unexpectedly fast at about 150 kDa from a 110 kDa monomer (25,28), so DPP9 at 130 kDa and DPP8 at 150 kDa may be dimers whereas the faster bands are monomer and possibly fragments (10,11).…”

Reversible Inactivation of Human Dipeptidyl Peptidases 8 and 9 by Oxidation

“…Identification of the four purified enzymes was confirmed by Edman N-terminal degradation at the Australian Proteome Analysis Facility. The DPIV enzyme activity of DPIV, DP8, DP9 and FAP was measured using H-AlaPro-pNA in the DPIV assay described previously [21]. [24] and a multiple sequence alignment of fifty proteins of the prolyl oligopeptidase gene family [25].…”