Chloroquine reduces arylsulphatase B activity and increases chondroitin-4-sulphate: implications for mechanisms of action and resistance

<p>Abstract</p> <p>Background</p> <p>The receptors for adhesion of <it>Plasmodium falciparum</it>-infected red blood cells (RBC) in the placenta have been identified as chondroitin-4-sulphate (C4S) proteoglycans, and the more sulphate-rich chondroitin oligosaccharides have been reported to inhibit adhesion. Since the anti-malarial drug chloroquine accumulates in lysosomes and alters normal lysosomal processes, the effects of chloroquine on the lysosomal enzyme arylsulphatase B (ASB, N-acetylgalactosamine-4-sulphatase), which removes 4-sulphate groups from chondroitin-4-sulphate, were addressed. The underlying hypothesis derived from the recognized impairment of attachment of parasite-infected erythrocytes in the placenta, when chondroitin-4-sulphation was increased. If chloroquine reduced ASB activity, leading to increased chondroitin-4-sulphation, it was hypothesized that the anti-malarial mechanism of chloroquine might derive, at least in part, from suppression of ASB.</p> <p>Methods</p> <p>Experimental methods involved cell culture of human placental, bronchial epithelial, and cerebrovascular cells, and the <it>in vitro </it>exposure of the cells to chloroquine at increasing concentrations and durations. Measurements of arylsulphatase B enzymatic activity, total sulphated glycosaminoglycans (sGAG), and chondroitin-4-sulphate (C4S) were performed using <it>in vitro </it>assays, following exposure to chloroquine and in untreated cell preparations. Fluorescent immunostaining of ASB was performed to determine the effect of chloroquine on cellular ASB content and localization. Mass spectrometry and high performance liquid chromatography were performed to document and to quantify the changes in chondroitin disaccharides following chloroquine exposure.</p> <p>Results</p> <p>In the human placental, bronchial epithelial, and cerebrovascular cells, exposure to increasing concentrations of chloroquine was associated with reduced ASB activity and with increased concentrations of sGAG, largely attributable to increased C4S. The study data demonstrated: 1) decline in ASB activity following chloroquine exposure; 2) inverse correlation between ASB activity and C4S content; 3) increased content of chondroitin-4-sulphate disaccharides following chloroquine exposure; and 4) decline in extent of chloroquine-induced ASB reduction with lower baseline ASB activity. Confocal microscopy demonstrated the presence of ASB along the cell periphery, indicating extra-lysosomal localization.</p> <p>Conclusions</p> <p>The study data indicate that the therapeutic mechanism of chloroquine action may be attributable, at least in part, to reduction of ASB activity, leading to increased chondroitin-4-sulphation in human placental, bronchial epithelial, and cerebrovascular cells. In vivo, increased chondroitin-4-sulphation may reduce the attachment of <it>P. falciparum</it>-infected erythrocytes to human cells. Extra-lysosomal localization of ASB and reduced impact of chloroquine when baseline ASB activity is less suggest possible mechanisms of resistance to the effects of chloroquine.</p

Region-Specific Cell Membrane N-Glycome of Functional Mouse Brain Areas Revealed by nanoLC-MS Analysis.

N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell-cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with ∼120 N-glycan compositions in each region, revealing significant differences in "brain-type" glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the presence of a previously unknown brain glyco-architecture. The region-specific molecular glyco fingerprints identified here will enable a better understanding of the critical biological roles that N-glycans play in the specialized functional brain areas in health and disease

Proteoglycan 4 (lubricin) is a highly sialylated glycoprotein associated with cardiac valve damage in animal models of infective endocarditis.

Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of tooth surfaces and are generally associated with oral health, but can also cause infective endocarditis (IE). These species express "Siglec-like" adhesins that bind sialylated glycans on host glycoproteins, which can aid the formation of infected platelet-fibrin thrombi (vegetations) on cardiac valve surfaces. We previously determined that the ability of S. gordonii to bind sialyl T-antigen (sTa) increased pathogenicity, relative to recognition of sialylated core 2 O-glycan structures, in an animal model of IE. However, it is unclear when and where the sTa structure is displayed, and which sTa-modified host factors promote valve colonization. In this study, we identified sialylated glycoproteins in the aortic valve vegetations and plasma of rat and rabbit models of this disease. Glycoproteins that display sTa vs. core 2 O-glycan structures were identified by using recombinant forms of the streptococcal Siglec-like adhesins for lectin blotting and affinity capture, and the O-linked glycans were profiled by mass spectrometry. Proteoglycan 4 (PRG4), also known as lubricin, was a major carrier of sTa in the infected vegetations. Moreover, plasma PRG4 levels were significantly higher in animals with damaged or infected valves, as compared with healthy animals. The combined results demonstrate that, in addition to platelet GPIbα, PRG4 is a highly sialylated mucin-like glycoprotein found in aortic valve vegetations and may contribute to the persistence of oral streptococci in this protected endovascular niche. Moreover, plasma PRG4 could serve as a biomarker for endocardial injury and infection

Glycan-protein cross-linking mass spectrometry reveals sialic acid-mediated protein networks on cell surfaces.

A cross-linking method is developed to elucidate glycan-mediated interactions between membrane proteins through sialic acids. The method provides information on previously unknown extensive glycomic interactions on cell membranes. The vast majority of membrane proteins are glycosylated with complicated glycan structures attached to the polypeptide backbone. Glycan-protein interactions are fundamental elements in many cellular events. Although significant advances have been made to identify protein-protein interactions in living cells, only modest advances have been made on glycan-protein interactions. Mechanistic elucidation of glycan-protein interactions has thus far remained elusive. Therefore, we developed a cross-linking mass spectrometry (XL-MS) workflow to directly identify glycan-protein interactions on the cell membrane using liquid chromatography-mass spectrometry (LC-MS). This method involved incorporating azido groups on cell surface glycans through biosynthetic pathways, followed by treatment of cell cultures with a synthesized reagent, N-hydroxysuccinimide (NHS)-cyclooctyne, which allowed the cross-linking of the sialic acid azides on glycans with primary amines on polypeptide backbones. The coupled peptide-glycan-peptide pairs after cross-linking were identified using the latest techniques in glycoproteomic and glycomic analyses and bioinformatics software. With this approach, information on the site of glycosylation, the glycoform, the source protein, and the target protein of the cross-linked pair were obtained. Glycoprotein-protein interactions involving unique glycoforms on the PNT2 cell surface were identified using the optimized and validated method. We built the GPX network of the PNT2 cell line and further investigated the biological roles of different glycan structures within protein complexes. Furthermore, we were able to build glycoprotein-protein complex models for previously unexplored interactions. The method will advance our future understanding of the roles of glycans in protein complexes on the cell surface

Multi-glycomic analysis of spheroid glycocalyx differentiates 2- and 3-dimensional cell models.

A multi-glycomic method for characterizing the glycocalyx was employed to identify the difference between 2-dimensional (2D) and 3-dimensional (3D) culture models with two human colorectal cancer cell lines, HCT116 and HT29. 3D cell cultures are considered more representative of cancer due to their ability to mimic the microenvironment found in tumors. For this reason, they have become an important tool in cancer research. Cell-cell interactions increase in 3D models compared to 2D, indeed significant glycomic changes were observed for each cell line. Analyses included the N-glycome, O-glycome, glycolipidome, glycoproteome, and proteome providing the most extensive characterization of the glycocalyx between 3D and 2D thus far. The different glycoconjugates were affected in different ways. In the N-glycome, the 3D cells increased in high-mannose glycosylation and in core fucosylation. Glycolipids increased in sialylation. Specific glycoproteins were found to increase in the 3D cell, elucidating the pathways that are affected between the two models. The results show large structural and biological changes between the 2 models suggesting that the 2 are indeed very different potentially affecting individual outcomes in the study of diseases