Glycosylation of the major polar tube protein of Encephalitozoon cuniculi

Abstract

To infect their host cells the Microsporidia use a unique invasion organelle, the polar tube complex. During infection, the organism is injected into the host cell through the hollow polar tube formed during spore germination. Currently, three proteins, PTP1, PTP2, and PTP3 have been identified by immunological and molecular techniques as being components of this structure. Genomic data suggests that Microsporidia are capable of O-linked, but not N-linked glycosylation as a post-translational protein modification. Cells were infected with Encephalitozoon cunicuili, labeled with radioactive mannose or glucosamine, and the polar tube proteins were examined for glycosylation. PTP1 was clearly demonstrated to be mannosylated consistent with 0-glycosylation. In addition, it was evident that several other proteins were mannosylated, but no labeling was seen with glucosamine. The observed post-translational mannosylation of PTP1 may be involved in the functional properties of the polar tube, including its adherence to host cells during penetration.

Fig. 1

Analysis of glycosylation in E. cuniculi. a Incorporation of[2]-³H-Mannose. Lane 1 spore wall (DTT insoluble fraction as described in methods), lane 2 E. cuniculi spore lysate, and lane 3 PTP preparation (DTT soluble fraction as described in methods). b. Incorporation of [2]-³H-Glucosamine. Lane 1 RK13 (host cell) lysate, lane 2 E. cuniculi spore lysate, and lane 3 PTP preparation (DTT soluble fraction as described in materials and methods). After SDS-PAGE the gel was dried overnight and used for signal detection. The gel was imaged at −80°C for 15 days as described in the methods

Fig. 2

Analysis of EcPTP1 Glycosylation. a Immunoblots using anti-EcPTP1. EcPTP1 is clearly detected in both the E. cuniculi spore lysate (lane1) and the immunoprecipitated material (lane 2). Immunoprecipitation was done using anti-EcPTP1 as described in methods. EcPTP1 migrates at ~50 kDa. The EcPTP1 antiserum also recognized a diffuse band at ~35 kDa which may be due to proteolysis of EcPTP1 during sample preparation. b. Autoradiography of pull-down assay using [2]-³H-Mannose-labeled E. cuniculi. Lane 1 RK13 lysate (negative control), lane 2 immunoglobin control, lane 3 residual PTP preparation bound to anti-EcPTP1 beads after washing and elution, and lane 4 eluted PTP material from anti-EcPTP1 beads after washing as described in methods. After SDS-PAGE, the gel was dried overnight and used for signal detection. The gel was imaged at −80°C for 15 days as described in the methods. This demonstrates that PTP1 is labeled by [2]-³H-Mannose