Bernd Kübler

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.

Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3

In the circulation, most of the insulin‐like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF‐binding protein (IGFBP)‐3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF‐binding fraction separated from human plasma. IGF ligand blotting, cross‐linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP‐3 showed a 5‐fold higher affinity for IGF‐II than IGFBP‐3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP‐3 functions.

Decreased intracellular degradation of insulin-like growth factor binding protein-3 in cathepsin L-deficient fibroblasts

Proteolysis of insulin‐like growth factor binding proteins (IGFBPs) is the major mechanism of releasing IGFs from their IGFBP complexes. Analysis of fibroblasts deficient for the lysosomal cysteine protease cathepsin L (CTSL) revealed an accumulation of IGFBP‐3 in the medium which was due neither to alterations in IGFBP‐3 mRNA expression nor to extracellular IGFBP‐3 protease activity. Incubation of CTSL‐deficient fibroblasts with radiolabeled IGFBP‐3 followed by subcellular fractionation indicates that both intact and fragmented IGFBP‐3 accumulate transiently in endosomal and lysosomal fractions of CTSL‐deficient cells. This suggests the involvement of CTSL in the intracellular degradation of IGFBP‐3 representing a new mechanism to regulate the extracellular concentration of IGFBP‐3.

Isolation and characterization of circulating fragments of the insulin-like growth factor binding protein-3.

Proteolysis of insulin‐like growth factor binding protein‐3 (IGFBP‐3), the major carrier of IGFs in the circulation, is an essential mechanism to regulate IGF bioavailability. To analyze naturally occurring IGFBP‐3 fragments a peptide library established from human hemofiltrate was screened. Three IGFBP‐3 fragments were detected with apparent molecular masses of 34, 16, and 11 kDa. Mass spectrometric and sequence analysis identified the 16 and 11 kDa peptides as glycosylated and non‐glycosylated N‐terminal fragments spanning residues Gly1–Ala98 of IGFBP‐3. Both the circulating forms and those secreted from IGFBP‐31–98 overexpressing cells bound IGF. Additionally, two smaller fragments (IGFBP‐3139–157 and IGFBP‐3139–159) were identified in the hemofiltrate. The data indicate that proteolysis of circulating IGFBP‐3 occurs in the variable domain at residues alanine 98, phenylalanine 138, glutamine 157, and tyrosine 159.

Interaction of Insulin-like Growth Factor II (IGF-II) with Multiple Plasma Proteins HIGH AFFINITY BINDING OF PLASMINOGEN TO IGF-II AND IGF-BINDING PROTEIN-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of ≥150 kDa. To investigate molecular interactions between proteins involved in IGF·IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of ≥440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.

In vivo processed fragments of IGF binding protein-2 copurified with bioactive IGF-II

Proteolysis of insulin-like growth factor binding proteins (IGFBPs), the major carrier of insulin-like growth factors (IGFs) in the circulation, is an essential mechanism to regulate the bioavailability and half-live of IGFs. Screening for peptides in human hemofiltrate, stimulating the survival of PC-12 cells, resulted in the isolation of C-terminal IGFBP-2 fragments and intact IGF-II co-eluting during the chromatographic purification procedure. The IGFBP-2 fragments exhibited molecular masses of 12.7 and 12.9kDa and started with Gly169 and Gly167, respectively. The fragments were able to bind both IGFs. The stimulatory effect of the purified fraction on the survival of the PC-12 cells could be assigned exclusively to IGF-II, since it was abolished by the addition of neutralizing IGF-II antibodies. We suggest that in the circulation IGF-II is not only complexed with intact IGFBP but also with processed IGFBP-2 fragments not impairing the biological activity of IGF-II.

Multiple post-translational modifications of mouse insulin-like growth factor binding protein-6 expressed in epithelial Madin–Darby canine kidney cells

Insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins (IGFBPs) participate in the regulation of proliferation and differentiation of epithelial cells. Expression of the growth-inhibitory murine IGFBP-6 in epithelial Madin-Darby canine kidney (MDCK) cells followed by 2D analysis revealed the presence of multiple isoforms. Metabolic labelling experiments showed that several IGFBP-6 isoforms are modified by phosphate and sulfate groups. Expression analysis of mutant IGFBP-6 further demonstrated that serine residue 143 is O-glycosylated. Substitution of serine 143 by alanine did slightly reduce the preferential sorting of mIGFBP-6 to the apical site in MDCK cells grown on semipermeable filters. Both the presence of multiple and heterogeneously modified isoforms of murine IGFBP-6 in MDCK cells, and the preferential secretion of non-glycosylated IGFBP-6 mutants to the apical side suggest that the major apical sorting signal is the protein moiety.