Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis

Glycoconj J. 2009 Aug;26(6):675-89. doi: 10.1007/s10719-008-9223-8. Epub 2009 Jan 29.

Abstract

We report the discovery and characterization of a glycosylated bacterial ABC-type phosphate transporter isolated from the peripheral blood mononuclear cell (PBMC) fraction of patients with visceral leishmaniasis (VL). Three disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) of 19, 56 and 65 kDa, respectively, had been identified and their purity, apparent mass and pI established by SDS-PAGE and isoelectric focusing. Western blot analyses showed that the 9-O-acetylated sialic acid is linked via alpha2-->6 linkage to a subterminal N-acetylgalactosamine. For the 56 kDa protein, N- as well as O-glycosylations were demonstrated by specific glycosidase treatment and found to account for more than 9 kDa of the protein mass. The presence of sialic acids was further confirmed through thin layer chromatography, fluorimetric HPLC and electrospray ionization-mass spectrometry. The protein was identified by mass spectrometry and de novo sequencing of five tryptic fragments as a periplasmic ABC-type phosphate transporter of Pseudomonas aeruginosa. The amino acid sequences of the assigned peptides had 83-100% identity with the NCBI entry for a Pseudomonas transporter protein. Based on the recently reported X-ray structure of a human phosphate-binding protein, we predicted a 3D structural model for the 56 kDa protein using homology and threading methods. The most probable N- and O-glycosylation sites were identified by combinations of sequence motif-searching bioinformatics tools, solvent accessibility calculations, structural environment analyses and mass spectrometric data. This is the first reported glycosylation as well as sialylation of the periplasmic component of an ABC-type phosphate transporter protein and of one of few identified bacterial glycoproteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / isolation & purification*
  • Child
  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • Flow Cytometry
  • Humans
  • Leishmaniasis, Visceral / blood*
  • Male
  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / isolation & purification*
  • Middle Aged
  • Molecular Weight
  • Sialoglycoproteins / chemistry*
  • Sialoglycoproteins / isolation & purification*
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Young Adult

Substances

  • Bacterial Proteins
  • Membrane Transport Proteins
  • Sialoglycoproteins