Recombinant Antibodies with Unique Specificities Allow for Sensitive and Specific Detection of Uncarboxylated Osteocalcin in Human Circulation

Calcified Tissue International https://doi.org/10.1007/s00223-020-00746-8 ORIGINAL RESEARCH Recombinant Antibodies with Unique Specificities Allow for Sensitive and Specific Detection of Uncarboxylated Osteocalcin in Human Circulation Milja Arponen1 · Eeva‑Christine Brockmann2 · Riku Kiviranta1 · Urpo Lamminmäki2 · Kaisa K. Ivaska1 Received: 8 May 2020 / Accepted: 5 August 2020 © The Author(s) 2020 Abstract Osteocalcin is a bone-specific protein which contains three glutamic acid residues (Glu) that undergo post-translational gamma-carboxylation. Uncarboxylated osteocalcin (ucOC) may participate in the regulation of glucose metabolism, thus measurement of ucOC could be useful in evaluating interactions between bone and glucose metabolism. We developed recombinant antibodies and immunoassay to specifically detect ucOC in human blood samples. ucOC-specific recombinant antibodies were selected from an antibody library by phage display. Four candidates were characterized, and one (Fab-AP13) was used to set up an immunoassay with a pre-existing MAb. Plasma ucOC levels were measured in subjects with normal fasting blood glucose (≤ 6 mmol/l, N = 46) or with hyperglycemia (≥ 7 mmol/l, N = 29). Further, we analyzed ucOC in age- and gender-matched patients with diagnosed type 2 diabetes (T2D, N = 49). Antibodies recognized ucOC without cross-reaction to carboxylated osteocalcin. Antibodies had unique binding sites at the carboxylation region, with Glu17 included in all epitopes. Immunoassay was set up and characterized. Immunoassay detected ucOC in serum and plasma, with on average 1.6-fold higher levels in plasma. ucOC concentrations were significantly lower in subjects with hyperglycemia (median 0.58 ng/ml, p = 0.008) or with T2D diagnosis (0.68 ng/ml, p = 0.015) than in subjects with normal blood glucose (1.01 ng/ ml). ucOC negatively correlated with fasting plasma glucose in subjects without T2D (r = − 0.24, p = 0.035) but not in T2D patients (p = 0.41). Our immunoassay, based on the novel recombinant antibody, allows for specific and sensitive detection of ucOC in human circulation. Correlation between ucOC and plasma glucose suggests interactions between osteocalcin and glucose metabolism in humans. Keywords Bone · Osteocalcin · Uncarboxylated osteocalcin · Glucose · Type 2 diabetes Introduction Bone is a metabolically active tissue that undergoes constant remodeling. Human osteocalcin is a small, 49 amino acid protein produced by osteoblasts. Osteocalcin undergoes post-translational modification, in which three glutamic Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00223-020-00746-8) contains supplementary material, which is available to authorized users. * Kaisa K. Ivaska 1 Institute of Biomedicine, University of Turku, 20520 Turku, Finland 2 Department of Biotechnology, University of Turku, Turku, Finland acid (Glu) residues, in positions 17, 21, and 24, convert to γ-carboxyglutamic acid (Gla) residues. This carboxylation process is vitamin K-dependent. Carboxylated osteocalcin (cOC) has a high affinity for Ca2+ and therefore it binds to bone mineral, whereas uncarboxylated (ucOC) form is unable to bind [1]. Both cOC and ucOC forms can be found in the circulation and circulating osteocalcin (total osteocalcin, tOC) consists also of truncated fragments and also partially carboxylated, or undercarboxylated forms [2, 3]. In humans, osteocalcin is incompletely carboxylated and uncarboxylation is more common at Glu17 than at two other positions [4, 5]. tOC measured from circulation is usually considered as a surrogate marker for bone formation [6]. In addition to biosynthesis in the osteoblasts, osteocalcin is also released from bone matrix during bone resorption [7] and thus, osteocalcin in circulation most likely reflects overall bone turnover. 13 Vol.:(0123456789) M. Arponen et al. Apart from being a marker for bone turnover, osteocalcin has been shown to participate as an endocrine factor in glucose and lipid metabolism. Osteocalcin-deficient mice have higher bone mineral density [8] but also increased body weight and impaired glucose metabolism [9]. Further studies indicated that the metabolic effect is due to ucOC form of osteocalcin, which has been shown to induce the secretion of insulin in pancreatic beta cells, both in mice [10] and in humans [11], adiponectin in adipocytes and to improve glucose uptake in skeletal muscle [9, 10, 12]. In particular, uncarboxylation at the first Glu residue (Glu17 in humans) has been linked to the endocrine effect in mice [13] and in humans [14]. The metabolic effect of ucOC is suggested to be mediated via G-protein-coupled receptor, GPRC6A [15, 16]. In humans, tOC levels have been shown to associate with circulating glucose levels, insulin sensitivity, and adiponectin concentration [17–20] in cross-sectional and observational studies which support the hypothesis of osteocalcin having an endocrine function. Meta-analyses have confirmed that tOC levels are lower in patients with type 2 diabetes (T2D) and that low tOC is a risk factor for T2D [21, 22]. On the contrary, Schwartz et al. reported no correlation between the incidence of diabetes and the use of antiresorptive therapy, which reduces circulating osteocalcin [23]. The association between the suggested hormonal form ucOC and glucose metabolism is less well understood. Low ucOC concentration has been shown to associate with T2D diagnosis [24]. Higher concentrations of ucOC are associated with enhanced insulin sensitivity and beta-cell function [25] but also correlate with better insulin secretion [26] as well as glycemic control and lower fasting plasma [27] in patients with diabetes. Other studies, however, have not been able to demonstrate a connection between ucOC and T2D [28] nor with insulin sensitivity [29] or insulin resistance [29, 30]. The evaluation of ucOC levels in human circulation has been done with different analytical methods, including direct ELISA [14] and hydroxyapatite binding assay [31], thus a reliable standardized method for measuring ucOC levels in circulation would be useful. Recombinant antibody phage libraries provide a rapid alternative to immunization-based hybridoma technology for development of new monospecific antibodies for virtually any kinds of antigens. As antibody libraries are selected in vitro, the reaction conditions are easily adjusted to direct the selections, for example, towards specific posttranslational modification sites [32]. In the current study, we developed recombinant antibodies that specifically detect ucOC form of osteocalcin, by using a synthetic single-chain fragment (scFv) antibody library [33]. We then developed a sensitive novel assay for the measurement of circulating ucOC in human blood samples and measured ucOC levels 13 in carefully selected samples to get further insight to the association of ucOC and glucose metabolism in humans. Materials and Me (...truncated)