Glypican-1 as a Biomarker for Prostate Cancer: Isolation and Characterization

Abstract

Prostate cancer is the most frequently diagnosed male visceral cancer and the second leading cause of cancer death in the United States. Standard tests such as prostate-specific antigen (PSA) measurement have poor specificity (33%) resulting in a high number of false positive reports. Consequently there is a need for new biomarkers to address this problem. The MIL-38 antibody was first described nearly thirty years ago, however, until now, the identification of the target antigen remained elusive. By a series of molecular techniques and mass spectrometry, the MIL-38 antigen was identified to be the highly glycosylated proteoglycan Glypican-1 (GPC-1). This protein is present in two forms; a membrane bound core protein of 55-60 kDa and secreted soluble forms of 40 kDa and 52 kDa. GPC-1 identification was confirmed by immuno-precipitation, western blots and ELISA. An ELISA platform is currently being developed to assess the levels of GPC-1 in normal, benign prostatic hyperplasia (BPH) and prostate cancer patients to determine whether secreted GPC-1 may represent a clinically relevant biomarker for prostate cancer diagnosis.

Keywords: Glypican 1; Prostate Cancer; Proteoglycan; Theranostic.

Figure 1

Flow cytometric analysis of MIL-38 binding to the surface antigen on DU-145 and C3 cells. The secondary antibody only control (DU-145 (dash) and C3 (dotted) cells) represents the baseline of the analysis. DU-145 cells show strong staining and C3 cells represent a negative population sample.

Figure 2

A. Western Blot of DU-145 membrane protein extracts treated with increasing concentrations of DTT. DU-145 (Lane 1) and C3 (Lane 2) are positive and negative controls respectively. B. Western Blot of immuno-precipitated of DU-145 membrane protein extracts (Lane A). The solid arrow indicates the immuno-precipitated antigen and the outlined arrow indicates the MIL-38 antibody. Antigen is not immuno-precipitated in Lanes (B) representing protein extracts with unconjugated beads or Lane (C) which represents antibody conjugated beads with no protein extracts. C. Western blot of membrane protein extracts from prostate cancer and normal prostate samples. The prostate cancer sample shows strong reactivity with MIL-38 at thirty times more expression than normal prostate samples.

Figure 3

Western blot of MIL-38 antigen secreted into conditioned serum free medium by DU-145 cells. Two proteins of sizes 40kDa and 52kDa were identified. DU-145 membrane protein extracts (MPEK) immuno-precipitates were run as controls and show the 55-60kDa antigen.

Figure 4

Mass spectrometric identification of the MIL-38 antigen (GPC-1). Identified amino acids are in bold (78% coverage). Amino acids identified in the secreted form are highlighted in grey.

Figure 5

Western blot of immuno-precipitates (IP) of MIL-38 or rabbit anti-GPC-1 conjugated beads with DU145 or C3 protein lysates (MPEK). DU145 protein lysates were immuno-precipitated by MIL-38 (A, C) and rabbit anti-GPC-1 (B) and probed by MIL-38 (A, B) or rabbit anti-GPC-1 (C). C3 protein lysates were not immuno-precipitated by either antibody. FT (flow through) is defined as the proteins in the supernatant not captured by the conjugated beads.

Figure 6

Two dimensional western blot using MIL-38 and rabbit anti-GPC-1 on DU-145 membrane protein extracts. The antigen identified by both antibodies overlap at 60kDa at isoelectric point 6 (indicated by ellipse).

Figure 7

Western blot of recombinant GPC-1. Both MIL-38 and rabbit anti-GPC-1 recognize the same non reduced forms of the protein. Rabbit anti-GPC-1 can also recognize the reduced form of the protein. R* indicates that the sample was processed under reducing conditions.

Figure 8

Bar graph showing various ELISA combinations of MIL-38 and rabbit anti-GPC-1. Recombinant GPC-1 was used as the analyte. All ELISA combinations show recombinant GPC-1 can be detected in a sandwich ELISA by MIL-38 as a capture antibody (A, C) and detector antibody (B, C).