Novel insulin-like growth factor-binding protein proteases : detection and characterization

Members of the insulin-like growth factor (IGF) system are known to have an impact on cell differentiation, proliferation and survival, and to be important regulators for nutrient metabolism. The IGF-binding proteins (IGFBP-1 to -6) control the availability of IGFs (IGF-I and IGF-II) to their receptors. In the circulation IGFs and IGFBP-3 or IGFBP-5 can bind an acid-labile subunit (ALS) to form a high molecular mass complex, which constitutes a circulating reservoir of IGFs. Post-translational modification and binding to cells and extracellular matrix may affect the IGF-dependent effects of IGFBPs. For example highly phosphorylated IGFBP-1, which is increased in catabolic conditions, has a high affinity for, and inhibits the actions of IGF-I. IGFBPs also have IGF-independent effects, through the interaction with cell surface structure or extracellular molecules. For example, the RGD sequence in the C-terminus of IGFBP-1 can interact with a cell surface á5â1 integrin to regulate cell migration and apoptosis. Proteolysis is another important posttranslational modification that affects IGFBP function at the target tissue.

The aims of this thesis are to characterise two novel IGFBP protease activities: (i) an IGFBP-3 protease activity detected in the thyroid of male mice and (ii) an IGFBP-1-specific protease activity discovered in human urine. In normal mice there is gender dimorphism in the circulating IGF system, with higher fasting IGF-I and IGFBP-3 ternary complex in male animals. IGFBP-3 protease activity was found in trunk blood from male mice and not females. This protease activity was due to tissue contamination likely to derive from thyroid. IGFBP-3 protease activity is present in the thyroid extracts from male mice. This protease has the characteristics of a calcium-activated serine protease and resulted in an 8-fold reduction in the IGF-I affinity of IGFBP-3.

We discovered a novel and specific IGFBP-1 protease activity that we purified from the urine of a patient with multiple myeloma and an inflammatory skin disorder. The proteolytic fraction contained azurocidin, which is a protease homologue hitherto considered inactive. The activity was abolished by an azurocidin monoclonal antibody. This protease efficiently cleaves both phosphorylated and nonphosphorylated IGFBP-1 at Ile130-Ser131 to generate fragments that, on BIAcore analysis, have higher association and dissociation rates than the intact IGFBP-1. In addition to having reduced affinity for IGFs, after cleavage of IGFBP-1. there was also a higher capacity for IGF-I binding, suggesting that both N- and C-terminal fragments may interact with ligand independently. Cleavage of IGFBP-1 decreased the inhibitory effect of IGF-II effect on MCF-7 breast cancer cell growth and on glucose uptake into cultures of human skeletal myotubes. Alone, proteolysed IGFBP-1. stimulated glucose uptake in muscle. No IGFBP-1. protease activity was detected in scrum from the patient; however there was evidence of immunoreactive fragments in the circulation.

In conclusion, novel IGFBP proteases may have important tissue-specific roles in determining the IGF-dependent and independent actions of IGFBPs.

List of scientific papers

I. Lewitt MS, Brismar K, Wang J, Wivall-Helleryd IL, Sindelar P, Gonzalez FJ, Bergman T, Bobek GA (2001). Responses of insulin-like growth factor (IGF)-I and IGF-binding proteins to nutritional status in peroxisome proliferator-activated receptor-alpha knockout mice. Growth Horm IGF Res. 11(5): 303-13.
https://doi.org/10.1054/ghir.2001.0247

II. Wang J, Grunler J, Lewitt MS (2004). Gender-specific pattern of insulin-like growth factor-binding protein-3 protease activity in mouse thyroid. Endocrinology. 145(3): 1137-43.
https://doi.org/10.1210/en.2003-1378

III. Wang J, Shafqat J, Hall K, Stahlberg M, Wivall-Helleryd IL, Bouzakri K, Zierath JR, Brismar K, Jornvall H, Lewitt MS. (2006). Specific cleavage of insulin-like growth factor-binding protein-1 by a novel protease activity. Cell Mol Life Sci. [Accepted]
https://doi.org/10.1007/s00018-006-6248-7

IV. Wang J, Brandt K, Wivall-Helleryd IL, Horn HV, Hall, K, Lewitt MS (2006). Detection of IGFBP-1 fragments in clinical samples. [Manuscript]