Osteopontin is cleaved at multiple sites close to its integrin-binding motifs in milk and is a novel substrate for plasmin and cathepsin D

Abstract

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg-Gly-Asp(145) sequence and were generated by cleavage of the Leu(151)-Arg(152), Arg(152)-Ser(153), Ser(153)-Lys(154), Lys(154)-Ser(155), Ser(155)-Lys(156), Lys(156)-Lys(157), or Phe(158)-Arg(159) peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg(152)-Ser(153) matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys(154)-Ser(155). Another endogenous milk protease, cathepsin D, cleaved the Leu(151)-Arg(152) bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the alpha(V)beta(3)- or alpha(5)beta(1)-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.

FIGURE 1.

Analysis of human milk OPN. A, Western blot analysis using a polyclonal OPN antibody. Lane 1, molecular mass standards. Lane 2, skim milk. Lane 3, OPN purified from milk. Lane 4, fraction I from B. Lane 5, fraction II from B. B, RP-HPLC of purified human milk OPN. Separation was performed on a Vydac C₁₈ column connected to a GE Healthcare system by stepwise increasing concentrations of 75% propan-2-ol in 0.1% trifluoroacetic acid (TFA) (dashed line) at a flow rate of 0.85 ml/min. Proteins were monitored at 226 nm (solid line).

FIGURE 2.

Reversed-phase HPLC separation and MS analysis of N-terminal OPN digested with Asp-N in a buffer containing 50% H₂¹⁸O. A, peptides were separated on a narrow-bore reversed-phase chromatography C₂/C₁₈ PC 2.1/10 column operated by a SMART system (GE Healthcare). Separation was carried out in 0.1% trifluoroacetic acid (TFA), and peptides were eluted with a gradient of 60% acetonitrile in 0.1% trifluoroacetic acid (dashed line) at a flow rate of 0.15 ml/min. The peptides were detected in the effluent by measuring the absorbance at 214 nm (solid line). B, all fractions were analyzed by MS, and representative data from fractions F1 to F8 are shown. Only the relevant m/z region is shown. In fractions F1 and F8, two internal peptides are identified based on the characteristic isotopic pattern caused by incorporation of ¹⁸O. Peptides present in fractions F2–F7 were identified as C-terminal peptides as these retain their natural isotopic distribution. C, schematic representation of OPN showing the identified cleavage sites in milk OPN. The location of phosphorylations and O-glycosylations is based on the post-translational modification pattern from human milk OPN (31). The RGD sequence is shown in boldface, and the cryptic integrin-binding site SVVYGLR is underlined. The cleavage sites identified in fraction F2–F7 are shown with arrows and are all located in the near vicinity of the integrin-binding motifs.

FIGURE 3.

Identification of cleavage products of OPN purified from human urine. OPN was purified from urine, and the isolated protein batch consists of both OPN fragments as well as the full-length protein. The urinary OPN forms were digested with Asp-N in a buffer containing 50% H₂¹⁸O and analyzed by MS without prior separation of the resulting peptides. Two peptides corresponding to Asp¹⁴⁵–Arg¹⁵² and Asp¹⁴⁵–Lys¹⁵⁴ were identified as C-terminal peptides, whereas other OPN peptides were observed with the monoisotopic pattern characteristic of internal peptides.

FIGURE 4.

OPN cleavage by thrombin, plasmin, and cathepsin D. Western blot analysis using a polyclonal OPN antibody. Lane 1, molecular weight standards. Lane 2, full-length OPN purified from milk (fraction II in Fig. 1B). Lane 3, N-terminal OPN fragments purified from human milk (fraction I in Fig. 1B). Lane 4–6, full-length OPN digested with thrombin (lane 4), plasmin (lane 5), and cathepsin D (lane 6).

FIGURE 5.

Plasmin, thrombin, and cathepsin D cleavage sites in OPN. A, cleavage sites for plasmin (P), thrombin (T), and cathepsin D (CD) are indicated with black arrows. Previously identified cleavage sites for MMPs (15) are shown with gray arrows. The RGD motif is highlighted in boldface, and the SVVYGLR sequence is underlined. B, alignment of mammalian OPN sequences. The region containing the identified cleavage sites in human milk is boxed. The integrin-binding motifs RGD and SVVYGLR are highlighted in gray. Glycosylation sites identified in human milk OPN (31) are indicated with a diamond, and corresponding residues in other OPN species are highlighted in black.

FIGURE 6.

Adhesion of cells to cleaved OPN. Adhesion assays were performed with two different cell types to surfaces coated with 5 pmol of full-length OPN or protease-cleaved human milk. OPN is referred to as OPN (full-length), nOPN (mixture of purified N-terminal fragments), OPN+PL (OPN cleaved with plasmin), OPN+TH (OPN cleaved with thrombin), and OPN+CD (OPN cleaved with cathepsin D). Bars show mean values for three wells per protein ± S.D. Data shown are representative of three independent experiments for each cell line. Adhesion to 1% bovine serum albumin was used as background and was subtracted from all values. A, adhesion of MDA-MB-435 cells to surfaces coated with the specified OPN forms. B, adhesion of MDA-MB-435 cells to wells coated with OPN after preincubation in the presence or absence of an antibody against the α_Vβ₃-integrin or an isotype IgG control (5 μg/ml). C, adhesion of K562 cells to surfaces coated with the specified OPN forms. D, adhesion of K562 cells to wells coated with OPN after preincubation in the presence or absence of an antibody against the α₅β₁-integrin or an isotype IgG control (5 μg/ml).