Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications

Abstract

OPN (osteopontin) is an integrin-binding highly phosphorylated glycoprotein, recognized as a key molecule in a multitude of biological processes such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation and cell survival. A significant regulation of OPN function is mediated through PTM (post-translational modification). Using a combination of Edman degradation and MS analyses, we have characterized the complete phosphorylation and glycosylation pattern of native human OPN. A total of 36 phosphoresidues have been localized in the sequence of OPN. There are 29 phosphorylations (Ser8, Ser10, Ser11, Ser46, Ser47, Thr50, Ser60, Ser62, Ser65, Ser83, Ser86, Ser89, Ser92, Ser104, Ser110, Ser113, Thr169, Ser179, Ser208, Ser218, Ser238, Ser247, Ser254, Ser259, Ser264, Ser275, Ser287, Ser292 and Ser294) located in the target sequence of MGCK (mammary gland casein kinase) also known as the Golgi kinase (S/T-X-E/S(P)/D). Six phosphorylations (Ser101, Ser107, Ser175, Ser199, Ser212 and Ser251) are located in the target sequence of CKII (casein kinase II) [S-X-X-E/S(P)/D] and a single phosphorylation, Ser203, is not positioned in the motif of either MGCK or CKII. The 36 phosphoresidues represent the maximal degree of modification since variability at many sites was seen. Five threonine residues are O-glycosylated (Thr118, Thr122, Thr127, Thr131 and Thr136) and two potential sites for N-glycosylation (Asn63 and Asn90) are not occupied in human milk OPN. The phosphorylations are arranged in clusters of three to five phosphoresidues and the regions containing the glycosylations and the RGD (Arg-Gly-Asp) integrin-binding sequence are devoid of phosphorylations. Knowledge about the positions and nature of PTMs in OPN will allow a rational experimental design of functional studies aimed at understanding the structural and functional interdependences in diverse biological processes in which OPN is a key molecule.

Figure 1. RP-HPLC separation of trypsin and thermolysin digests of OPN

(A) RP-HPLC of tryptic peptides. Peptides were separated on a μRPC C₂/C₁₈ PC 3.2/3 column operated by a Pharmacia SMART system. Separation was carried out in 0.1% TFA and peptides were eluted with a gradient of 60% acetonitrile in 0.1% TFA (- - -) at a flow rate of 0.15 ml/min. The peptides were detected in the effluent by measuring the absorbance at 214 nm (——). (B) RP-HPLC of thermolytic peptides. Peptides were separated by RP-HPLC on a Vydac C₁₈ column operated by a Pharmacia LKB system. Separation was carried out in 0.1% TFA and eluted with a gradient of 80% acetonitrile in 0.1% TFA (- - -) at a flow rate of 0.85 ml/min. The peptides were detected in the effluent by measuring the absorbance at 226 nm (——).

Figure 2. Localization of PTMs in human OPN

Solid lines indicate characterized peptides. P denotes identified phosphorylation. ◆, glycosylations. The two putative N-glycosylation sites and the integrin receptor-binding RGD sequence are highlighted. Peptides are named with reference to the elution profiles in Figure 1.

Figure 3. MALDI–TOF-MS analysis of Th11 from Figure 1(B)

The protonated mass at m/z 1591.58 corresponds to the triple phosphorylated peptide T⁵⁷LPSKSNESHDH⁶⁸. The characteristic fragmentation pattern confirms the presence of three phosphorylations in the peptide.

Figure 4. MALDI–TOF-MS analysis of intact native human OPN

The average mass peak at 42.9 kDa represents native human OPN. The peaks at approx. 22 and 85 kDa represent the derived (M2H⁺) and (2MH⁺) protonated species respectively.

Figure 5. Alignment of human and bovine OPN sequences

Modified residues are highlighted and introduced gaps are indicated by broken lines. P denotes identified phosphorylation and filled diamonds symbolize glycosylations.