Bones provide support and protection for organs in the body. However, over the last 15 years researchers have discovered that bones also release chemicals known as hormones, which can travel to other parts of the body and cause an effect. The cells responsible for making bone, known as osteoblasts, produce a hormone called osteocalcin which communicates with a number of different organs, including the pancreas and brain.

When osteocalcin reaches the pancreas, it promotes the release of another hormone called insulin which helps regulate the levels of sugar in the blood. Osteocalcin also travels to other organs such as muscle, where it helps to degrade fats and sugars that can be converted into energy. It also has beneficial effects on the brain, and has been shown to aid memory and reduce depression.

Osteocalcin has largely been studied in mice where levels are five to ten times higher than in humans. But it is unclear why this difference exists or how it alters the role of osteocalcin in humans. To answer this question, Al Rifai et al. used a range of experimental techniques to compare the structure and activity of osteocalcin in mice and humans.

The experiments showed that mouse osteocalcin has a group of sugars attached to its protein structure, which prevent the hormone from being degraded by an enzyme in the blood. Human osteocalcin has a slightly different protein sequence and is therefore unable to bind to this sugar group. As a result, the osteocalcin molecules in humans are less stable and cannot last as long in the blood. Al Rifai et al. showed that when human osteocalcin was modified so the sugar group could attach, the hormone was able to stick around for much longer and reach higher levels when added to blood in the laboratory.

These findings show how osteocalcin differs between human and mice. Understanding this difference is important as the effects of osteocalcin mean this hormone can be used to treat diabetes and brain disorders. Furthermore, the results reveal how the stability of osteocalcin could be improved in humans, which could potentially enhance its therapeutic effect.

Osteocalcin (OCN) is a peptide hormone secreted by osteoblasts, the bone forming cells (Lee et al., 2007). It regulates glucose metabolism by promoting beta cell proliferation and insulin secretion, and by improving insulin sensitivity (Pi et al., 2011; Ferron et al., 2012). In addition to its role in the regulation of energy metabolism, OCN is also involved in male fertility by promoting testosterone synthesis by Leydig cells (Oury et al., 2011), in muscle adaptation to exercise by improving glucose and fatty acid uptake in myocytes (Mera et al., 2016a), and in acute stress response through the inhibition of post-synaptic parasympathetic neurons (Berger et al., 2019). Overall, OCN might acts as an ‘anti-geronic’ circulating factor preventing age-related cognitive decline and muscle wasting (Khrimian et al., 2017; Mera et al., 2016b; Oury et al., 2013b). The G protein coupled receptor family C group six member A (GPRC6A) mediates OCN function in beta cells, muscles and testis (Mera et al., 2016a; Pi et al., 2011; Oury et al., 2013a), while the G protein coupled receptor 158 (Gpr158) mediates its function in the brain (Khrimian et al., 2017; Kosmidis et al., 2018).

Within the bone tissue, OCN undergoes a series of post-translational modifications (PTM) that are critical for the regulation of its endocrine functions. Prior to its secretion, in the osteoblast endoplasmic reticulum, the OCN precursor (pro-OCN) is γ-carboxylated on three glutamic acid residues (Glu) by the vitamin K-dependent γ-glutamyl carboxylase (Ferron et al., 2015). In the trans-Golgi network, pro-OCN is next cleaved by the proprotein convertase furin releasing mature carboxylated OCN (Gla-OCN) (Al Rifai et al., 2017). The presence of the negatively charged Gla residues allows Gla-OCN to bind hydroxyapatite, the mineral component of the bone extracellular matrix (ECM). It is during bone resorption that Gla-OCN is decarboxylated through a non-enzymatic process involving the acidic pH generated by the osteoclasts, ultimately leading to the release of bioactive uncarboxylated OCN (ucOCN) in the circulation (Ferron et al., 2010a; Lacombe et al., 2013). The conclusion that ucOCN represents the bioactive form of this protein in rodents is supported by cell-based assays, mouse genetics and in vivo studies [reviewed in Mera et al., 2018].

The role of OCN in the regulation of glucose metabolism appears to be conserved in humans. Human ucOCN can bind and activate human GPRC6A (De Toni et al., 2016) and promotes beta cell proliferation and insulin synthesis in human islets (Sabek et al., 2015), while mutations or polymorphisms in human GPRC6A are associated with insulin resistance (Di Nisio et al., 2017; Oury et al., 2013a). Finally, several cross-sectional and observational studies have detected a negative association between OCN or ucOCN, and insulin resistance or the risk of developing type 2 diabetes in various human populations (Lin et al., 2018; Turcotte et al., 2020; Lacombe et al., 2020).

Yet, some important species divergences exist between mice and humans with regard to OCN biology. First, only 30 out of the 46 amino acids (i.e. 65%) composing mature mouse OCN are conserved in human OCN. This is in striking contrast with other peptide hormones involved in the control of energy metabolism such as leptin and insulin whose respective sequence display about 85% conservation between mouse and human. Second, the circulating concentrations of OCN, even though decreasing with age in both species, are five to ten times higher in mice than in humans throughout life span [(Mera et al., 2016a) see also Table 1]. Based on these observations, we hypothesized that the post-translational regulation of OCN may be different between these two species, resulting in increased mouse OCN half-life in circulation.

Here, using proteomics and cell-based assays, we identified O-glycosylation as a novel PTM presents in mouse OCN, and showed that this modification increases mouse OCN half-life in plasma ex vivo and in vivo. In contrast, mature human OCN does not contain the O-glycosylation site found in the mouse protein and consequently is not normally glycosylated. Yet, a single point mutation in human OCN is sufficient to elicit its O-glycosylation and to increase its half-life in plasma.

In this study we identified O-glycosylation as a novel PTM regulating mouse ucOCN half-life in the circulation. We also showed that O-glycosylation is not found on human mature OCN, but that O-glycosylation of human OCN by means of a single amino acid change can improve its half-life in plasma ex vivo. These findings reveal an important species difference in the regulation of OCN and may also have important implication for the future use of recombinant ucOCN as a therapeutic agent in humans.

Numerous secreted proteins are subjected to mucin-type (GalNAc-type) O-glycosylation, a PTM which is initiated in the Golgi apparatus and involves multiple sequential glycosylation steps to produce diverse O-glycan structures (Steentoft et al., 2013; Bennett et al., 2012). The initiation of O-glycosylation is catalyzed by the GalNAc-Ts isoenzymes, however, the specific protein sequence(s) targeted by each of the GalNAc-Ts remain poorly characterized, although weak acceptor motifs for these have been identified by in vitro analyses (Perrine et al., 2009; Gerken et al., 2006). Here, we demonstrate that mouse OCN is O-glycosylated likely on serine 8, which is located within the amino acid sequence SVPSPDP¹¹. Interestingly, this sequence strongly matches the consensus site previously defined for GalNAc-T1 and GalNAc-T2 (Gerken et al., 2006). In particular, the presence of proline residues in position −one, +one and +three has been shown to be determinant in the recognition of peptide substrates by GalNAc-T1 and GalNAc-T2 in vitro. We used an isogenic cell library with combinatorial engineering of isoenzyme families to explore the regulation of osteocalcin O-glycosylation by GalNAc-Ts (Narimatsu et al., 2019a; Narimatsu et al., 2019b). Combined knock out of GalNAc-T1, 2 and 3 only partially abolishes mouse OCN O-glycosylation in HEK293 cells, suggesting that OCN may be a substrate for additional GalNAc-Ts.

O-glycosylation was shown to interfere with the action of proprotein convertases on several pro-hormones and receptors (Goth et al., 2015; Kato et al., 2006; May et al., 2003; Schjoldager and Clausen, 2012; Schjoldager et al., 2010; Goth et al., 2017). This appears not to be the case for OCN as its O-glycosylation does not interfere with its processing by furin in vitro and in vivo. In other proteins, such as leptin and erythropoietin, glycosylation adducts were shown to increase protein stability and half-life in circulation (Elliott et al., 2003; Creus et al., 2001). More recently, O-glycosylation was shown to protect atrial natriuretic peptide from proteolytic degradation by neprilysin or insulin-degrading enzyme in vitro (Hansen et al., 2019). Additional studies showed that sialic acid residues present in the glycosylation adducts increase protein charge, thereby improving serum half-life and decreasing liver and renal clearance (Morell et al., 1971; Runkel et al., 1998; Perlman et al., 2003; Ziltener et al., 1994). Although our data suggest that protection from proteolytic cleavage by plasmin in the plasma might be one mechanism by which O-glycosylation extend OCN half-life, we cannot exclude that in vivo, O-glycosylation may also decrease liver and renal clearance of OCN.

Mouse OCN contains arginine (R) residues at position 16 and 40, corresponding to R20 and R44 in human OCN, which could be potential cleavage sites for plasmin (Figure 5A). A putative plasmin cleavage site is present in mouse (i.e. AYK↓R⁴⁰, where the arrow indicates the cleavage site) and human (i.e. AYR↓R⁴⁴) OCN at the corresponding position (Backes et al., 2000). Bovine OCN, like the human protein, does not possess the O-glycosylation site found in mouse, and was shown to be cleaved by plasmin in vitro between R43 and R44 (Novak et al., 1997). Here we show that plasmin is sufficient to reduce the concentration non-glycosylated ucOCN, by more than 90% in two hours, confirming that mouse ucOCN is also a plasmin substrate. Supporting a role for plasmin in the cleavage of mouse OCN in vivo, OCN serum levels are decreased in mice deficient in plasmin activator inhibitor I (PAI-I), which have higher plasmin activity (Tamura et al., 2013). Interestingly, circulating plasmin activity increases with age in rats and humans (Paczek et al., 2009; Paczek et al., 2008), an observation which could in part explain the gradual reduction of serum OCN concentrations during aging (Mera et al., 2016a). Notably, a nonsynonymous rare variant (rs34702397) in OCN resulting in the conversion of R43 in a glutamine (Q) exists in humans of African ancestry and was nominally associated with insulin sensitivity index and glucose disposal in a small cohort of African Americans (Das et al., 2010). Since the R43Q variant eliminates the potential plasmin cleavage site in the C-terminal region, it will be interesting to further investigate if subjects carrying this variant have higher OCN circulating levels.

We also show that O-glycosylation protects mouse ucOCN from degradation in INS-1 cell cultures and consequently increases its biological activity. Interestingly, non-glycosylated ucOCN is stable when incubated in the INS-1 cell culture media at 37°C without cells (data not shown), suggesting that the proteolytic activity originates from the INS-1 cells and not from the culture media. The identity of a putative β-cell-derived protease responsible for ucOCN degradation remains unknown, since plasmin is expressed exclusively by the hepatocytes. Nevertheless, these results suggest that it may be more appropriate to use glycosylated ucOCN when studying the function of mouse OCN in cell culture.

We found that human OCN is not subjected to O-glycosylation and that consequently it has a reduced half-life in plasma ex vivo. The O-glycosylation sequence ‘SVPSPDP¹¹’ of mouse OCN is conserved in the human protein, except for the amino acids corresponding to S5 and S8, which are replaced by a proline (P9) and a tyrosine (Y12) (i.e. ‘PVPYPDP¹⁵’). Remarkably, we could introduce O-glycosylation into human OCN by a single amino acid change (Y12S). O-glycosylated human OCN is protected from degradation in plasma ex vivo similarly to the glycosylated mouse protein. Hence, this difference in the O-glycosylation status of OCN could potentially explain why circulating level of OCN in 1- to 6-month-old mice is 5–10 times higher than the level measured in young or adult human (Table 1).

It remains unknown if the increased half-life of O-glycosylated ucOCN will result in improved biological activity in vivo, although it was shown to be the case for other proteins (Baudys et al., 1995; Runkel et al., 1998; Elliott et al., 2003). Our cell-based assay does suggest that O-glycosylated ucOCN is active, at least on β-cells. We also showed that mouse OCN is endogenously O-glycosylated in vivo and that more than 80% of OCN, including the ucOCN fraction, is O-glycosylated in osteoblast supernatant and in serum. This suggests that the bioactive form of OCN is O-glycosylated in vivo in mice.

In summary, this work identified O-glycosylation as a previously unrecognized OCN PTM regulating its half-life in circulation in mice. This modification is not conserved in human, yet introducing O-glycosylation in human ucOCN also increases its half-life in plasma. These findings reveal an important difference between mouse and human OCN biology and also provide an approach to increase recombinant human OCN half-life in vivo, which might be relevant for the future development of OCN-based therapies for human diseases.

All the numerical data and the original western blots are available in the source data Excel file submitted with the manuscript. The raw proteomics data have been uploaded to a public server.

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Author details

1. Omar Al Rifai

   1. Molecular Physiology Research unit, Institut de Recherches Cliniques de Montréal, Montréal, Canada
   2. Programme de biologie moléculaire, Université de Montréal, Montréal, Canada

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

2. Catherine Julien

   Molecular Physiology Research unit, Institut de Recherches Cliniques de Montréal, Montréal, Canada

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Julie Lacombe

   Molecular Physiology Research unit, Institut de Recherches Cliniques de Montréal, Montréal, Canada

   Contribution

   Conceptualization, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Denis Faubert

   Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, Canada

   Contribution

   Resources, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Erandi Lira-Navarrete

   University of Copenhagen, Faculty of Health Sciences, Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Copenhagen, Denmark

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Yoshiki Narimatsu

   University of Copenhagen, Faculty of Health Sciences, Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Copenhagen, Denmark

   Contribution

   Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

7. Henrik Clausen

   University of Copenhagen, Faculty of Health Sciences, Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Copenhagen, Denmark

   Contribution

   Resources, Supervision, Writing - review and editing

   Competing interests

   No competing interests declared

8. Mathieu Ferron

   1. Molecular Physiology Research unit, Institut de Recherches Cliniques de Montréal, Montréal, Canada
   2. Programme de biologie moléculaire, Université de Montréal, Montréal, Canada
   3. Département de Médecine, Université de Montréal, Montréal, Canada
   4. Division of Experimental Medicine, McGill University, Montréal, Canada

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   mathieu.ferron@ircm.qc.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5858-2686

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