Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis.
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.
Ghoshal
Angana
A
Department of Infectious Disease and Immunology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata, 700032, India.
Mukhopadhyay
Sumi
S
Demine
Rodion
R
Forgber
Michael
M
Jarmalavicius
Saulius
S
Saha
Bibhuti
B
Sundar
Shyam
S
Walden
Peter
P
Mandal
Chhabinath
C
Mandal
Chitra
C
D000328:Adult; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002648:Child; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005434:Flow Cytometry; D006801:Humans; D007898:Leishmaniasis, Visceral / Q000097:blood*; D008297:Male; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D008875:Middle Aged; D008970:Molecular Weight; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055815:Young Adult
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D012795:Sialoglycoproteins