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

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.

9-O-Acetylated Sialoglycoproteins Are Important Immunomodulators in Indian Visceral Leishmaniasis

“…Furthermore, these antibodies, as reported earlier, are leishmanicidal in activity; that is, they are capable of triggering the classical complement pathway, thereby causing Leishmania cell death (5). Alternatively, several other 9-OAcSGPs have been documented on immune cells (65, 56, and 19 kDa) and erythrocytes (112, 107, 103, 57, 51, and 48 kDa) of VL patients (4,11,22). Since the alteration of sialic acid expression (via the alteration of enzymes responsible for sialic acid metabolism) has been well documented as a disease-induced phenomenon (26,48,49), the induction of these 9-OAcSGPs on the host cells of patients with active VL is possibly a result of parasitic infection.…”

“…Since the alteration of sialic acid expression (via the alteration of enzymes responsible for sialic acid metabolism) has been well documented as a disease-induced phenomenon (26,48,49), the induction of these 9-OAcSGPs on the host cells of patients with active VL is possibly a result of parasitic infection. Importantly, previous studies have demonstrated that each of these 9-O-AcSGP molecules is different from the others at the protein level, as revealed in isoelectric-focusing studies by the presence of discrete bands indicating their molecular heterogeneity, even though they are all alike in having 9-O-AcSA on their terminal surface (22). Thus, anti-9-O-AcSGP antibodies driven by the 9-OAcSGPs present on the parasites recognize the diseaseassociated 9-O-AcSGPs on PBMC VL via recognition of the terminal 9-O-AcSA.…”

“…9-OAcSGPs on PBMC VL were affinity purified using achatinin-H as an affinity matrix as described elsewhere (22). The binding of 9-O-AcSGPs to achatinin-H was detected by incubation with anti-9-O-AcSGP antibodies (1 g) that were affinity purified from sera of patients with active VL overnight at 4°C (10).…”

“…c The experiment was performed with the same group of active VL patients used for the previous study, as described elsewhere (22). After successful chemotherapy, paired samples from this group of patients were used for comparison.…”

“…In this regard, we have demonstrated the presence of disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on different immune cells of peripheral blood mononuclear cells (PBMC) of patients with active VL (PBMC VL ) using a lectin, achatinin-H, having preferential affinity toward terminal 9-O-AcSA derivatives ␣2-6 linked to subterminal N-acetylgalactosamine, 9-O-AcSA␣2-6GalNAc (4,22,37,38). However, their role in modulating the immune response and thereby regulating the clinical course of the disease remained unaddressed.…”