Relative deficiency of acidic isoforms of osteopontin from stone former urine

Abstract

We have tested the relative electrophoretic mobility of osteopontin (OPN) isolated from urine obtained from normal individuals (NU) against similar samples derived from the urine of stone formers (SFU) using high-resolution isoelectric focusing (isoelectric point, pI range 3.5-4.5) in 2D electrophoresis, with Western blot detection. We also report the results from competitive ELISA analyses of these samples. We demonstrated that human urinary OPN has a discrete four band separation pattern that conforms to four previously documented OPN isoforms. The lower two M(r) isoforms migrate to a greater degree toward the acidic end of the gel than do the higher two M(r) isoforms. Densitometry of the signal reveals significant difference in the migration pattern of OPN from SFU as compared to that from NU based on an analysis of the spot intensities grouped in 0.1 pI unit increments. A novel method for the calculation of a weight-averaged pI based on the relative signal strength in an OPN 2D Western blot was developed. The analysis revealed a significantly increased weight-averaged pI values for the higher M(r) forms of OPN in the stone former compared to normal population. Additionally, alkaline phosphatase-treated NU samples resulted in a significant average pI shift of 0.05 units in the alkaline direction, suggesting that a decrease in the average degree of phosphorylation could be responsible for the difference between NU and SFU pI.

Fig. 1

Human OPN detection in a standard Western blot from both NU and SFU

Fig. 2

2D human OPN Western detection of representative NU and SFU samples run on 3.5–4.5 pI IEF strips. No signal was evident greater than a pI of 4.2. *Greater proportion of OPN 1 and 2 signal in the 3.5–3.7 range for NU versus SFU; 45 versus 10%, P = 0.03 (highlighted in the large ovals). ^ŦLesser proportion of OPN 3 and 4 signal in the 3.7–3.8 range for NU versus SFU; 10 versus 18%, P = 0.05 (highlighted with the small ovals)

Fig. 3

Bar graphs depicting the average percent signal per 0.1 pI increment for total OPN, OPN 1 and 2, and OPN 3 and 4. Total OPN signal (panel A) is significantly lower in SFU versus NU for the 3.6 region (£ = 0.04). The combined OPN 1 and 2 signal (panel B) is significantly lower in SFU versus NU for the 3.5 and 3.6 regions (* = 0.03). The combined OPN 3 and 4 signal (panel C) is significantly higher in SFU versus NU for the 3.7 region (Ŧ=0.05). Standard error bars are shown