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Dipeptidyl peptidase IV-related protein (DPPX) was found to be preferentially expressed in the brain tissue. We isolated two rat cDNA clones encoding DPPX-S and DPPX-L from a brain cDNA library, of which DPPX-L had a longer sequence at the NH2 terminus. The biosynthesis of DPPXs was examined in both in vitro and in vivo systems. In the cell-free translation system, DPPX-S and DPPX-L were synthesized as 93-kDa and 97-kDa forms, respectively, which are in good agreement with the molecular masses estimated from their primary structure. In COS-1 cells transfected with the cDNAs, DPPX-S and DPPX-L were initially synthesized as 113-kDa and 117-kDa forms, respectively, with high-mannose type oligosaccharides, which were then converted to 115-kDa and 120-kDa forms, mostly with the complex-type sugar chains. Immunofluorescence-micro-scopic observations confirmed that both DPPXs were expressed on the cell surface. DPPXs were found to have no enzyme activity of DPPIV, even when they were mutated to have the consensus active-site sequence Gly-X-Ser-X-Gly for serine proteases. Immu-noblot analysis of samples prepared from various rat tissues demonstrated that DPPX-S, but not DPPX-L, was detectable only in the brain tissue. These results indicate that, of the two isoforms, DPPX-S is preferentially expressed in the brain tissue as the surface glycoprotein without protease activity, although its function remains unknown at present. [ABSTRACT FROM AUTHOR]

We have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses (∼80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218↑Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162↑Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H]ethanolamine, a component of glycosylphospha-tidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162→Thr) molecule caused by its aggregation is the molecular basis for the lethal hypophosphatasia carrying this mutation. [ABSTRACT FROM AUTHOR]