Glycoconjugate Journal

, Volume 26, Issue 6, pp 675–689

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

  • Angana Ghoshal
  • Sumi Mukhopadhyay
  • Rodion Demine
  • Michael Forgber
  • Saulius Jarmalavicius
  • Bibhuti Saha
  • Shyam Sundar
  • Peter Walden
  • Chhabinath Mandal
  • Chitra Mandal
Article

DOI: 10.1007/s10719-008-9223-8

Cite this article as:
Ghoshal, A., Mukhopadhyay, S., Demine, R. et al. Glycoconj J (2009) 26: 675. doi:10.1007/s10719-008-9223-8

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 α2→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.

Keywords

Pseudomonas aeruginosa Sialylated ABC-type phosphate transporter protein De novo peptide sequencing Molecular modeling Visceral leishmaniasis 

Abbreviations

9-O-AcSA

9-O-acetylated sialic acid

9-O-AcSGP

9-O-acetylated sialoglycoprotein

ABC

ATP-binding cassette

BSA

bovine serum albumin

BSM

bovine submandibular mucin

Da

dalton

DIG

digoxigenin

DSA

Datura stramonium agglutinin

DMB

1,2-diamino-4,5-methylenedioxybenzene

ddH2O

double distilled water

EDTA

ethylenediaminetetraacetic acid

ELISA

enzyme-linked immunosorbent assays

ESI-MS

electrospray ionization mass spectrometry

FITC

fluorescein isothiocyanate

GalNAc

N-acetylgalactosamine

GNA

Galanthus nivalis agglutinin

GP-56

56 kDa O-acetylated sialoglycoprotein

GT

glycosyltransferase

HPLC

high-performance liquid chromatography

HRP

horseradish peroxidase

HPBP

human phosphate-binding protein

IEF

isoelectric focusing

kDa

kilodalton

LD

Leishmania donovani

MAA

Maackia amurensis agglutinin

MALDI-TOF MS

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Mab

monoclonal antibody

MS

mass spectrometry

GlcNAc

N-acetylglucosamine

Neu5Ac

N-acetylneuraminic acid

OAcSGP

O-acetylated sialoglycoproteins

PA

Pseudomonas aeruginosa

PB

potassium phosphate buffer

PBMC

peripheral blood mononuclear cells

PBS

phosphate-buffered saline

PDB

protein data base

PE

phycoerythrin

PMF

peptide mass fingerprint

PMSF

phenylmethylsulfonyl fluoride

PNA

peanut agglutinin

PSD

post-source decay

RIA

radioimmunoassay

RMS

root mean square

RMSD

root mean square deviation

SA

sialic acid

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SNA

Sambucus nigra agglutinin

TBS

Tris-buffered saline

TLC

thin layer chromatography

TM

transmembrane

VL

visceral leishmaniasis

Supplementary material

10719_2008_9223_MOESM1_ESM.pdf (1.2 mb)
ESM Figure S1Identification of GP-56 by mass spectrometry (PDF 1.16 MB)

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Angana Ghoshal
    • 1
  • Sumi Mukhopadhyay
    • 1
  • Rodion Demine
    • 3
  • Michael Forgber
    • 3
  • Saulius Jarmalavicius
    • 3
  • Bibhuti Saha
    • 4
  • Shyam Sundar
    • 5
  • Peter Walden
    • 3
  • Chhabinath Mandal
    • 2
  • Chitra Mandal
    • 1
    • 6
  1. 1.Department of Infectious Disease and ImmunologyIndian Institute of Chemical BiologyKolkataIndia
  2. 2.Structural Biology and Bioinformatics DivisionIndian Institute of Chemical BiologyKolkataIndia
  3. 3.Department of DermatologyCharité-Universitätsmedizin Berlin, Humboldt UniversityBerlinGermany
  4. 4.Department of Tropical MedicineSchool of Tropical MedicineKolkataIndia
  5. 5.Department of Medicine, Institute of Medical SciencesBanaras Hindu UniversityVaranasiIndia
  6. 6.Infectious Disease and Immunology DivisionIndian Institute of Chemical BiologyKolkataIndia

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