Proteolysis of insulin-like growth factors (IGF) and IGF binding proteins by cathepsin D.

Various proteinases have been postulated to function in limited proteolysis of insulin-like growth factor binding proteins (IGFBPs) contributing to the regulation of IGF bioavailability. In this study, we report on the in vitro degradation of IGFs and IGFBPs by the purified acidic aspartylprotease cathepsin D that has been shown to proteolyze IGFBP-3. Recombinant human [125I] IGFBP-1 to -5 were processed by cathepsin D to fragments of defined sizes in a concentration dependent manner, whereas IGFBP-6 was not degraded. Ligand blotting revealed that none of the IGFBP-1 or -3 fragments formed by cathepsin D retain their ability to bind IGF. By N-terminal sequence analysis of nonglycosylated IGFBP-3 fragments produced by cathepsin D, at least four different cleavage sites were identified. Some of these cleavage sites were identical or differed by one amino acid from sites used by other IGFBP proteases described. The IGFBP-3 and -4 cleavage sites produced by cathepsin D are located in the nonconserved central region. IGF-I and -II, but not the unrelated platelet-derived growth factor BB, were degraded by cathepsin D in a time and concentration-dependent manner. We speculate that the major functional site of cathepsin D is intracellular and may be involved 1) in the selected clearance either of IGFBP or IGFs via different endocytic pathways or 2) in the general lysosomal inactivation of the IGF system.

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.

Proteolysis of IGFBPs by cathepsin D in vitro and in cathepsin D-deficient mice.

Affinity-purified lysosomal protease cathepsin D cleaved recombinant human IGFBP-1 to -5 in fragments of defined sizes, while IGFBP-6 was not degraded. To assess the role of cathepsin D for proteolytic processing of IGFBP in vivo, serum from cathepsin D-deficient mice and conditioned media from cathepsin D-deficient fibroblasts and organ explants were analyzed. No differences for the pattern and level of IGFBPs were detected. When conditioned media from fibroblasts were incubated at acid pH, proteolysis of IGFBP-1 and -4 was observed only in media derived from cathepsin D-expressing cells. Additional experiments showed that the proteolysis of IGFBP-4 is mediated by cathepsin D and not by a protease activated by cathepsin D. The IGFBP-4 degrading activities in media from organ explants from cathepsin D-deficient mice were found to be sensitive to inhibitors of aspartyl and cysteine proteases. The data indicate that different classes of acid pH-dependent proteases can contribute to the regulation of IGFBP-4 abundance.

Glycosylation and phosphorylation of arylsulfatase A.

The glycosylation and phosphorylation of the lysosomal enzyme arylsulfatase A was analyzed by a combination of metabolic labeling, tryptic fragmentation, mass spectrometry, and radiosequencing. The results demonstrate that all three potential N-glycosylation sites at Asn residues 158, 184, and 350 are utilized in arylsufatase A and carry high mannose or hybride type oligosaccharides. Phosphorylation of mannose residues is restricted to oligosaccharides at the first and third N-glycosylation site (Asn-158 and Asn-350). Both are phosphorylated with comparable efficiency. An earlier study had shown that a mutant arylsulfatase A containing only the second N-glycosylation site at Asn-184 folds correctly and is phosphorylated (Gieselmann, V., Schmidt, B., and von Figura, K. (1992) J. Biol. Chem. 267, 13262-13266). The lack of phosphorylation at Asn-184 in wild type arylsulfatase A therefore indicates that in vivo the presence of oligosaccharides can interfere with phosphorylation of other sites or that phosphorylation occurs in an ordered manner whereby phosphorylation of one site can affect the phosphorylation of another site.