Identification and characterization of glycation adducts on osteocalcin

Abstract

Osteocalcin is an important extracellular matrix bone protein that contributes to the structural properties of bone through its interactions with hydroxyapatite mineral and with collagen I. This role may be affected by glycation, a labile modification the levels of which has been shown to correlate with bone fragility. Glycation starts with the spontaneous addition of a sugar onto a free amine group on a protein, forming an Amadori product, and then proceeds through several environment-dependent stages resulting in the formation of an advanced glycation end product. Here, we induce the first step of this modification on synthetic osteocalcin, and then use multiple mass spectrometry fragmentation techniques to determine the location of this modification. Collision-induced dissociation resulted in spectra dominated by neutral loss, and was unable to identify Amadori products. Electron-transfer dissociation showed that the Amadori product formed solely on osteocalcin's N-terminus. This suggests that the glycation of osteocalcin is unlikely to interfere with osteocalcin's interaction with hydroxyapatite. Instead, glycation may interfere with its interaction with collagen I or another bone protein, osteopontin. Potentially, the levels of glycated osteocalcin fragments released from bone during bone resorption could be used to assess bone quality, should the N-terminal fragments be targeted.

Keywords: Bone; Bone matrix; Glycation; Non-collagenous protein; Osteocalcin.

Figure 1

Osteocalcin sequence annotating areas of interest to this study, highlighting in boxes the potential glycation sites, the trypsin digestion sites by arrows. A shaded “E” signifies a carboxylation site, or a “Gla” residue.

Figure 2

Schematic of the formation of Amadori Products on a protein

Figure 3

Spectra 3A–C show the areas of interest from LC/MS runs of osteocalcin incubated with buffer alone, glucose, and ribose.

Figure 4

Spectra 4A–B show areas of interest from the LC/MS spectra of digested osteocalcin control and incubated with ribose.

Figure 5

Spectrum showing the presence of the OC 20–42 peptide present in both single and double carboxylations. Empty boxes show areas in the spectrum where peaks would be expected should the peptide possess an Amadori product, which are absent.

Figure 6

Spectrum (a) shows the fragmentation of the 773.7233 peak, which corresponds to the unmodified OC N-terminal peptide. (b) Shows the annotated spectrum of the 817.7371 peak fragmentation, which corresponds to the weight of the OC N-terminal peptide with a ribosylation. The fragmentation shows c- and z- ions indicating the presence of the ribosylation on the n-terminus of the peptide. Both spectra shown are derived from the ribosylated OC sample.