The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli Type 1 fimbriae

Abstract

Background: GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria.

Methods: An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding.

Results: GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues.

Conclusion: GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.

Figure 1

GP2 binding to bacteria with Type 1 fimbria. Silver stain of SDS-PAGE (a) and protein immunoblotting (b) of purified recombinant human GP2 protein. Protein immunoblotting was performed with rabbit anti-human GP2 antisera. SBTI, soybean trypsin inhibitor. (B) Binding assay of bacteria with (AAEC185/pSH2) and without (AAEC185) Type 1 fimbria performed in microtiter plates coated with 1 μg/well of GP2, uromodulin, or BSA protein. ** p < 0.01 compared with AAEC185. (C) Microtiter wells coated with GP2 or BSA proteins at concentrations of .1, .5, 1, 2 and 5 μg/ml, followed by incubation with AAEC185/pSH2 (1 × 10⁸ CFU) bacteria that express Type 1 fimbria. All assays were performed in triplicate. The CFU/well are calculated as the mean ± SD (n = 3) and the error bar represents 1 SD.

Figure 2

Specificity of GP2 binding to bacteria with Type 1 fimbria. (A) AAEC185/pSH2 bacteria binding assay with microtiter wells previously coated with 100 ng of GP2 protein followed by preincubation with either rabbit anti-human GP2 antiserum or control preimmune serum. (B) 5 μg of GP2 protein was preincubated with Type 1 fimbriated bacteria (AAEC185/pSH2) before application to the microtiter plates. Data are expressed as the mean CFU/well and the error bars equal 1SD. All assays were performed in triplicate. ** p < 0.01 compared with control preimmune serum (A) or without GP2 preincubation (B).

Figure 3

GP2 glycosylation with mannose residues is required for bacteria binding. (A) SDS-PAGE of GP2 and uromodulin (UMOD) proteins with (+) and without (-) deglycosylation with peptide:N-glycosidase F treatment. (B) Binding of Type 1 fimbriated bacteria (AAEC185/pSH2) to glycosylated and deglycosylated GP2 or UMOD. (C) AAEC185/pSH2 bacteria binding to GP2 after pre-incubation with 1% (v/v) α-D-mannose. Data are expressed as the mean CFU/well of triplicate samples and the error bar represents 1SD. ** p < 0.01 compared with untreated controls.

Figure 4

GP2 binding to bacteria with M₁H and M₁L Type 1 fimbriae. Different bacteria strains were tested for the ability to bind GP2 in the microtiter well binding assay. 1 × 10⁸ CFU/well bacteria were used for each assay. FimH designates bacteria with or without FimH containing Type 1 fimbria. Bound bacteria were expressed as the mean CFU/well and the error bar represents 1SD (n = 3). ** p < 0.01 compared to BSA.