INTRODUCTION. The use of mouse genetics recently highlighted coordination by endocrine regulation between bone, energy metabolism, and endogenous sex hormones. Osteocalcin, a bone matrix protein that regulates hydroxyapatite size and shape through its vitamin‑K-dependent γ‑carboxylated form, unraveled endocrinological functions in its circulating forms, that are not only simple markers of bone formation. Undercarboxylated osteocalcin administration regulates gene and protein expression in adipocytes, in pancreatic β cells, and in Leydig cells by a G protein-coupled receptor, GPRC6A receptor, with a potential gender selectivity. Total serum osteocalcin concentrations in humans are inversely associated with measures of glucose metabolism and cardiovascular risk factors; however, human data are inconclusive with regard to the role of uncarboxylated osteocalcin because most studies do not account for the influence of vitamin K or differentiate between the different γ‑carboxylated forms of osteocalcin. Intriguingly GPRC6A receptor mediates also the non-genomic effects of androgens and, although the information about SHBG-receptor structure is not conclusive, evidence seems to suggest that a G protein-coupled receptor is involved.
AIM of the STUDY. Starting from clinical observations, we investigated the protein-protein interaction computational predictors of osteocalcin and SHBG with GPRC6A receptor, and we validated experimentally in vitro in a two-step approach.
MATHERIAL and METHODS. Clinical and biochemical characteristics were studied in a cohort of 91 obese patients and healthy controls in a cross-sectional study. 3-D protein structure alignment analysis and protein docking analysis were resolved with a four (i.e. TM-align, FATCAT, TriangleMatch, TopMatch) and three (i.e. GRAMM-X, ZDOCK, PatchDock) different algorithms approach, respectively. ClickMD-min script was used as a molecular dynamic platform for computational analysis of the conformational structure of osteocalcin in presence or absence of Ca2+, developed with MOE software. Steered molecular dynamics simulations related to the carboxylation state were performed, and the data compared with the spectroscopic techniques results. Gene and protein expression analysis and cell-surface receptor binding assays (i.e. immunofluorescence and flow cytometry analysis) on HEK-293T cells experimentally validated the former results in vitro.
RESULTS. Our study shows for the first time the existence of the competition for a specific binding site between osteocalcin and SHBG on human cells expressing GPRC6A receptor. The results are supported by a computational prediction analysis, describing amino acid residues of SHBG from Gly145 to Leu161 as a highly predicted interface. Our experiments describe the structure of human osteocalcin in its carboxylated and undercarboxylated forms for the first time. The influence of the binding of calcium on osteocalcin structure seems to be stronger than the presence of γ-carboxylated glutamic acid residues. Our clinical data show an imbalance between the γ‑carboxylated forms of osteocalcin in a cohort of hypogonadic obese an overweight patients, with decreased levels of SHBG and altered levels of the other sex hormones.
CONCLUSIONS. The current two-step approach offers a target approach of investigation directly in humans, that has the potential to identify novel pathophysiological pathways as well as novel therapeutic possibilities. The computational basis of the possible binding of SHBG and osteocalcin has been experimentally validated and can directly lead to the synthesis of a peptide, whose physiological and therapeutic implications represent a feasible perspective for future research in an area of paramount importance, such as the cross-talk between bone, energy metabolism, and endogenous sex hormones
