Molecular characterization and gene disruption of mouse lysosomal putative serine carboxypeptidase 1

Abstract

The lysosomal compartment plays a pivotal role in the degradation of macromolecules within the cell. To date, over 60 soluble lysosomal hydrolases and accessory proteins and 25 lysosomal membrane proteins have been identified [1][2][3]. Defects in the lysosomal proteins mostly result in one of about 50 lysosomal storage diseases (LSDs) which are characterized by the accumulation of undigested materials in the lysosomes. As a result of the clinical relevance of soluble lysosomal proteins in LSDs and a notable number of LSD-like diseases of unknown etiology, there is a common interest in the identification of the proteome of the lysosomal compartment and of the soluble luminal lysosomal mannose 6-phosphate (M6P)-containing The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC) is a lysosomal matrix protein that was initially identified in a screen for genes induced by retinoic acid. Recently, it has been spotlighted by several proteome analyses of the lysosomal compartment, but its cellular function and properties remain unknown to date. In this study, Scpep1 from mice was analysed with regard to its intracellular processing into a mature dimer consisting of a 35 kDa N-terminal fragment and a so far unknown 18 kDa C-terminal fragment and the glycosylation status of the mature Scpep1 fragment. Although Scpep1 shares notable homology and a number of structural hallmarks with the well-described lysosomal carboxypeptidase protective protein ⁄ cathepsin A, the purified recombinant 55 kDa precursor and the homogenates of Scpep1-overexpressing cells do not show proteolytic activity or increased serine carboxypeptidase activity towards artificial serine carboxypeptidase substrates. Hence, we disrupted the Scpep1 gene in mice by a gene trap cassette, resulting in a Scpep1 ⁄ b-galactosidase ⁄ neomycin phosphotransferase fusion protein. The fusion protein is devoid of the C-terminal half of Scpep1, including two amino acids of the assumed catalytic triad which is indispensable for its predicted serine carboxypeptidase activity. However, Scpep1-deficient mice were viable and fertile, and did not exhibit either lysosomal storage or reduced lysosomal SC activity under any tested condition.Abbreviations AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; CBZ, benzyloxycarbonyl; Cpvl, carboxypeptidase vitellogenic-like; CPY, carboxypeptidase Y; Ctsa, protective protein ⁄ cathepsin A; FA, furylacryloyl; geo, b-galactosidase ⁄ neomycin phosphotransferase; Lamp1, lysosomal associated membrane protein 1; LSD, lysosomal storage disease; M6P, mannose 6-phosphate; MEFs, mouse embryonic fibroblasts; MPR, mannose 6-phosphate receptor; PNGase F, peptide N-glycosidase F; RISC, retinoid-inducible serine carboxypeptidase; SC, serine carboxypeptidase; Scpep1, serine carboxypeptidase 1; Scpep1-gt, Scpep1 gene trap.

Arylsulfatase G inactivation causes loss of heparan sulfate 3- O -sulfatase activity and mucopolysaccharidosis in mice

“…The tissues were prepared for histochemical and immunofluorescence staining by standard procedures. Tissue samples for TEM were prepared as described previously (27 shown at the glucuronic acid (third residue) is relatively rare, which agrees with the finding that no pentasulfated trisaccharides were found as NRE structures (Fig. 3A).…”

Functional Characterization of a Putative Serine Carboxypeptidase in Vascular Smooth Muscle Cells

“…6,7 A recent report also failed to reveal intrinsic protease activity for SCPEP1. 20 Nevertheless, we hypothesize that SCPEP1-induced increases in SMC growth and migration require catalytic activity because SCPEP1 S167A was ineffective in mediating these processes. Loss-of-function studies in vitro demonstrate that SCPEP1 is necessary for SMC growth and migration, a finding substantiated in vivo following vascular injury in Scpep1 KO mice.…”

Molecular Characterization of Arylsulfatase G

“…2B). Of note, only 10% of total protein from fractions F1 to F4 was loaded compared with fractions derived from differential centrifugation, demonstrating 30 -40-fold enrichment of the 34-kDa fragment in the F2 fraction, which is typically also observed for other lysosomal proteins in the tyloxapol-based fractionation experimental setup (21,25). Subcellular fractionation of liver derived from Arsg KO mice and subsequent immunoblot analysis confirmed specific detection of ARSG bands (Fig.…”

“…Tritosome Preparation-Tritosomes were prepared as described previously (21). In brief, mice were injected with tyloxapol (Sigma) 4 days prior to sacrifice.…”