Recently, we and others identified the 66.3-kDa protein as one of several putative novel lysosomal matrix proteins by analyzing mannose 6-phosphate receptors binding proteins [Kollmann K., Mutenda K.E., Balleininger M., Eckermann E., von Figura K., Schmidt B., Lübke T. (2005) Identification of novel lysosomal matrix proteins by proteome analysis. Proteomics 5(15), 3966-3678, Sleat D.E., Lackland H., Wang Y., Sohar I., Xiao G., Li H., Lobel P. (2005) The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins. Proteomics. 5(6), 1520-1532]. Here, we describe the expression of the mouse 66.3-kDa protein in HT1080 cells in which it is synthesized as a precursor of about 75 kDa and subsequently processed by limited proteolysis to mature polypeptides accumulating in the lysosomal compartment. The lysosomal localisation of the endogenous 66.3-kDa protein was verified by indirect immunofluorescence in mouse embryonic fibroblasts and by subcellular fractionation of tyloxapol-filled mouse liver lysosomes. Northern blot analysis reveals high transcriptional levels in testis, liver and kidney, whereas Western blot analysis shows high protein levels in brain, heart, lung and spleen. Interestingly, in mouse the endogenous 66.3-kDa protein is processed in a highly tissue-dependent manner to mature forms.

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.

The 66.3 kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75 kDa preproprotein which is further processed into 28 and 40 kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3 kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3 kDa variant and the double-chain form consisting of a 28 kDa fragment and a 40 kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3 kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9 A on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4 A using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an R(free) factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks.

Cell-type-Syrian-hamster-kidney-21 BSA Rinderserumalbumin bzw. beziehungsweise °C Grad Celsius C. familiaris Canis familiaris Cat-D Cathepsin D cDNA komplementäre DNA Ci Curie (2,22 # 10 6 counts per minute) UZ Ultrazentrifuge V Volt V/cm Volt/Zentimeter v/v Volumen zu Volumen w/v Gewicht zu Volumen Y2H Yeast-two-Hybrid YPDA Yeast peptone dextrose adenine z. B. zum Beispiel Chemische Elemente wurden mit den üblichen Buchstaben abgekürzt. Die Aminosäuren wurden entweder im Drei-oder Einbuchstabencode angegeben. Bei einigen Begriffen wurden die englischen Fachtermini benutzt, weil auch in der deutschsprachigen Fachliteratur eine Übersetzung dieser Begriffe unüblich und unzureichend ist.