Further characterization of the target of a potential aptamer biomarker for pancreatic cancer: cyclophilin B and its posttranslational modifications

Abstract

Posttranslational modifications on proteins can serve as useful biomarkers for disease. However, their discovery and detection in biological fluids is challenging. Aptamers are oligonucleotide ligands that demonstrate high affinity toward their target proteins and can discriminate closely related proteins with superb specificity. Previously, we generated a cyclophilin B aptamer (M9-5) that could discriminate sera from pancreatic cancer patients and healthy volunteers with high specificity and sensitivity. In our present work we further characterize the aptamer and the target protein, cyclophilin B, and demonstrate that the aptamer could discriminate between cyclophilin B expressed in human cells versus bacteria. Using mass-spectrometric analysis, we discovered post-translational modifications on cyclophilin B that might be responsible for the M9-5 selectivity. The ability to distinguish between forms of the same protein with differing post-translational modifications is an important advantage of aptamers as tools for identification and detection of biomarkers.

FIG. 1.

Cyclophilin B aptamer M9-5 directly binds to the purified recombinant cyclophilin B (CypB) expressed in mammalian (HEK293) cells. (A) Western blot analysis of purified recombinant CypB protein expressed in bacterial (Escherichia coli) and mammalian (HEK293) cells by using CypB antibody revealed polypeptide bands migrating at ∼20 kD. MiaPaCa-2 secretome was used as the positive control. (B) The radiolabeled M9-5 was incubated with the purified recombinant CypB protein expressed in bacterial (E.coli) and mammalian (HEK293) cells and the binding affinity of M9-5 for the proteins were tested by using the radioactive filter-binding assay. M9-5 bound the CypB expressed in HEK293 cells with a dissociation constant (K_d) of ∼50 nM but demonstrated no affinity for the CypB protein expressed in the E. coli cells. M9-5 demonstrated no binding affinity for two polyhistidine-tagged proteins, GRP78 (glucose-regulated protein, 78 kD) and PRDX-1 (peroxiredoxin-1), used as controls.

FIG. 2.

Secreted cyclophilin B is non-glycosylated. (A) Recombinant CypB proteins (5 μg/lane) expressed in bacterial (E. coli) and mammalian (HEK293) cells were resolved in a 4%–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to the glycostaining protocol. Horseradish peroxidase (HRP, 5 μg), a known glycosylated protein, was used as a positive control and soybean trypsin inhibitor (5 μg), a non-glycosylated protein was used as a negative control for the assay. Only the glycoprotein HRP was stained and was detected as a magenta-colored polypeptide band by the glycostaining (upper panel). The same gel was stained with Coomassie blue for loading control (lower panel). (B) MiaPaCa-2 secretome was clarified by using either a wheat germ agglutinin (WGA)-agarose or a concanavalin A (ConA)-agarose column. The eluate and flow-through (FT) were collected; the buffer was exchanged with buffer F and was subjected to the subsequent assays. (C) Equal volume (20 μL/lane) of the eluate and FT from the WGA-agarose and ConA-agarose columns were resolved in a 4%–20% SDS-PAGE. A number of different polypeptide bands were detected in each fraction when the gel was subjected to the silver staining protocol. (D) Equal volume (10 μL/lane) of the eluate and FT from the WGA-agarose and ConA-agarose columns and the input were resolved in a 4%–20% SDS-PAGE and subjected to western blot by using CypB antibody. Polypeptide bands migrating at ∼20 kD were detected in the input and FT fractions of both the WGA-agarose and ConA-agarose columns. No detectable bands were visualized in the eluate fractions of either WGA-agarose or ConA-agarose columns indicating that CypB does not bind to these lectin columns. Purified recombinant CypB (0.01 μg) was used as the positive control. (E) The eluate and FT fractions of WGA-agarose and ConA-agarose columns were tested for M9-5 binding activity by using the radioactive filter-binding assay. The M9-5 binding activity was present only in the flow-through fractions of both the WGA-agarose and ConA-agarose columns. No binding activity was observed in the eluate fractions of WGA-agarose and ConA-agarose columns.

FIG. 3.

Purification of CypB from the MiaPaCa-2 secretome for the mass-spectrometric analysis and posttranslational modification search. (A) Schema for the purification of CypB from the MiaPaCa-2 secretome. (B) The MiaPaCa-2 secretome, WGA-agarose column FT, and eluate were resolved in a 4%–20% SDS-PAGE and subjected to the silver staining protocol. A polypeptide band (indicated by the arrow) with similar migration pattern as CypB was detected at ∼20 kD in the WGA-FT fraction. No bands were visualized in the same migration zone of the corresponding WGA-eluate fraction. (C) Equal volume (10 μL/lane) of different fractions from the CypB purification steps; MiaPaCa-2 secretome, S-column eluate, S-column eluate (dialyzed), WGA-agarose column flow-through, and WGA-Agarose column eluate were resolved in a 4%–20% SDS-PAGE and subjected to western blot by using CypB antibody. Polypeptide bands migrating at ∼20 kD were detected in all the fractions except in the WGA-agarose eluate lane. Recombinant CypB (0.01 μg) was used as the positive control.