Expression and maturation of human cathepsin D in baby-hamster kidney cells

Abstract

In medium and in homogenates from baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA, an elevated activity of cathepsin D was found as compared to non-transfected cells. The elevated activity was removed by titrating the homogenates with an anti-(human cathepsin D) antibody. Metabolic labelling and immunoprecipitation revealed that, in the transfected cells, human cathepsin D was synthesized as a 53-kDa precursor indistinguishable from that found in human cells. A portion of the precursor was secreted and the remainder was processed to intermediate and mature chains within a few hours of synthesis. The precursor that was released from the transfected cells had a slightly smaller apparent size than that from cultured human fibroblasts. This difference was abrogated when the precursors were treated with glycopeptidase F. In the intracellular small chain a difference was observed in the size of carbohydrate chains that were cleavable with endo-,l-N-acetylglucosaminidase H. Sequence analysis of the N-termini of mature intracellular cathepsin D indicated a N-terminal trimming in both large and small chains from both human and transfected hamster cells. The proteolytic maturation of human cathepsin D in BHK cells closely resembles that in human cells, whereas a portion of the carbohydrate side chains is processed differently. The trimmingof the N-termini in mature cathepsin D is proposed to be a part of the maturation and aging of this protein. INTRODUCTIONHuman cathepsin D is a lysosomal pepstatin-sensitive aspartic proteinase consisting of two polypeptides [1]. It is synthesized as a high-molecular-mass (53 kDa) precursor that is subject to maturation upon segregation from the secretory pathway [2]. In a cell-free system using porcine cathepsin D, cDNA synthesis of a preprocathepsin D with an N-terminal signal sequence has been demonstrated [3]. The amino acid sequence of human preprocathepsin D has been deduced from the nucleotide sequence of the corresponding cDNA [4][5][6]. Its sequence is similar

The novel mouseconnexin39gene is expressed in developing striated muscle fibers

“…For stable transfection of HeLa cells, the mCx39 coding region was cut out of the pGEM-T easy vector with XhoI and EcoRI and cloned into the XhoI/EcoRI restricted multiple cloning site of the expression vector pMJgreen (J. Degen, unpublished results). This vector contained an AsnI/StuI fragment (2570 bp) derived from the pEGFP-N1 vector (Clontech, Palo Alto, CA, USA), which was cloned into the NotI/ClaI treated pBEHpac18 vector (~3700 bp) (Horst and Hasilik, 1991). The resulting expression plasmid was denoted pMJCx39.…”

Glycosylphosphatidylinositol Anchors of Membrane Glycoproteins Are Binding Determinants for the Channel-forming Toxin Aerolysin

“…The plasmid construct of the chimeric GPI-anchored form of human cathepsin D (CD; a gift from Dr. E. Ogier-Denis, Faculté de Médecine de Xavier Bichat, Paris) has been reported earlier (28). It contains the human CD cDNA (29) fused with the GPI anchor signal sequence of human 5Ј-nucleotidase (30). Plasmids were transfected into CHO-K1 cells for stable expression using a CaCl 2 precipitation method (31) or into COS cells for transient expression using a DEAE-dextran and chloroquine method (32).…”

Molecular Cloning and Functional Expression of the Mouse Gap Junction Gene Connexin-57 in Human HeLa Cells

“…From this plasmid, a KpnI/BamHI fragment containing the coding region of mouse Cx57 was cloned into the KpnI/BamHI site of the transfection vector pBEHpac18 (37), which contained the SV40 early promoter, a polyadenylation signal, and a gene that conferred resistance to puromycin.…”