scholarly journals An affinity chromatography and glycoproteomics workflow to profile the chondroitin sulfate proteoglycans that interact with malarial VAR2CSA in the placenta and in cancer

Glycobiology ◽  
2020 ◽  
Vol 30 (12) ◽  
pp. 989-1002 ◽  
Author(s):  
Alejandro Gómez Toledo ◽  
Jessica Pihl ◽  
Charlotte B Spliid ◽  
Andrea Persson ◽  
Jonas Nilsson ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Affinity Purification ◽  
Chondroitin Sulfate Proteoglycans ◽  
Intervillous Space ◽  
Core Proteins ◽  
Heterogenous Group ◽  
Tumor Tissues ◽  
Infected Cells ◽  
Cancer Tissues

Abstract Chondroitin sulfate (CS) is the placental receptor for the VAR2CSA malaria protein, expressed at the surface of infected erythrocytes during Plasmodium falciparum infection. Infected cells adhere to syncytiotrophoblasts or get trapped within the intervillous space by binding to a determinant in a 4-O-sulfated CS chains. However, the exact structure of these glycan sequences remains unclear. VAR2CSA-reactive CS is also expressed by tumor cells, making it an attractive target for cancer diagnosis and therapeutics. The identities of the proteoglycans carrying these modifications in placental and cancer tissues remain poorly characterized. This information is clinically relevant since presentation of the glycan chains may be mediated by novel core proteins or by a limited subset of established proteoglycans. To address this question, VAR2CSA-binding proteoglycans were affinity-purified from the human placenta, tumor tissues and cancer cells and analyzed through a specialized glycoproteomics workflow. We show that VAR2CSA-reactive CS chains associate with a heterogenous group of proteoglycans, including novel core proteins. Additionally, this work demonstrates how affinity purification in combination with glycoproteomics analysis can facilitate the characterization of CSPGs with distinct CS epitopes. A similar workflow can be applied to investigate the interaction of CSPGs with other CS binding lectins as well.

scholarly journals Expanding the Chondroitin Sulfate Glycoproteome — But How Far?

2021 ◽  
Vol 9 ◽  
Author(s):  
Fredrik Noborn ◽  
Mahnaz Nikpour ◽  
Andrea Persson ◽  
Jonas Nilsson ◽  
Göran Larson
Keyword(s):  
Animal Models ◽  
Chondroitin Sulfate ◽  
Structural Complexity ◽  
The Novel ◽  
Connective Tissues ◽  
Cellular Physiology ◽  
Core Proteins ◽  
Attachment Sites ◽  
Improved Methods

Chondroitin sulfate proteoglycans (CSPGs) are found at cell surfaces and in connective tissues, where they interact with a multitude of proteins involved in various pathophysiological processes. From a methodological perspective, the identification of CSPGs is challenging, as the identification requires the combined sequencing of specific core proteins, together with the characterization of the CS polysaccharide modification(s). According to the current notion of CSPGs, they are often considered in relation to a functional role in which a given proteoglycan regulates a specific function in cellular physiology. Recent advances in glycoproteomic methods have, however, enabled the identification of numerous novel chondroitin sulfate core proteins, and their glycosaminoglycan attachment sites, in humans and in various animal models. In addition, these methods have revealed unexpected structural complexity even in the linkage regions. These findings indicate that the number and structural complexity of CSPGs are much greater than previously perceived. In light of these findings, the prospect of finding additional CSPGs, using improved methods for structural and functional characterizations, and studying novel sample matrices in humans and in animal models is discussed. Further, as many of the novel CSPGs are found in low abundance and with not yet assigned functions, these findings may challenge the traditional notion of defining proteoglycans. Therefore, the concept of proteoglycans is considered, discussing whether “a proteoglycan” should be defined mainly on the basis of an assigned function or on the structural evidence of its existence.

scholarly journals Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts.

1989 ◽  
Vol 108 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
G David ◽  
V Lories ◽  
A Heremans ◽  
B Van der Schueren ◽  
J J Cassiman ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Human Lung ◽  
Core Protein ◽  
Human Fibroblasts ◽  
Chondroitin Sulfate Proteoglycans ◽  
Lung Fibroblasts ◽  
Hydrophobic Membrane ◽  
Human Lung Fibroblasts ◽  
Core Proteins

Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.

Characteristics of chondroitin sulfate proteoglycans of differentiating nonchondrogenic tissues

1983 ◽  
Vol 61 (12) ◽  
pp. 1251-1259 ◽  
Author(s):  
Terry Crawford
Keyword(s):  
Chondroitin Sulfate ◽  
Chondroitin Sulfate Proteoglycans ◽  
Chondroitin Sulfate Proteoglycan ◽  
Density Gradients ◽  
Embryonic Brain ◽  
Chick Embryos ◽  
Sulfate Proteoglycan ◽  
Major Class ◽  
Core Proteins ◽  
Form Part

The chondroitin 4- and 6-sulfate proteoglycan monomers from two differentiating tissues (brain and muscle of 14-day chick embryos) were compared by indirect means, including use of chondroitinase AC, with the major class of cartilage chondroitin sulfate proteoglycan monomers. In addition to slower sedimentation in sucrose density gradients, the monomers from both noncartilaginous tissues have longer chondroitin sulfate chains and exhibit little difference in the susceptibilities of their core proteins to papain and trypsin, in contrast to the cartilage monomer. However, slight differences in average sizes and polydispersity of fragments formed by enzyme treatments and β-elimination were found between the proteochondroitin sulfate monomers of embryonic brain and muscle. Although the chondroitin sulfate chains are attached to separate tryptic peptides from the majority of other sulfated glycosaminoglycans in the noncartilaginous tissues, it is still uncertain whether these units form part of separate proteoglycan monomers.

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta

1986 ◽  
Vol 883 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Junichiro Aikawa ◽  
Mamoru Isemura ◽  
Hiroshi Munakata ◽  
Noboru Ototani ◽  
Chie Kodama ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Thoracic Aorta ◽  
Chondroitin Sulfate Proteoglycans ◽  
Isolation And Characterization

scholarly journals Electron microscopic characterization of chick embryonic skeletal muscle proteoglycans.

1985 ◽  
Vol 100 (5) ◽  
pp. 1767-1776 ◽  
Author(s):  
D G Pechak ◽  
D A Carrino ◽  
A I Caplan
Keyword(s):  
Electron Microscopy ◽  
Chondroitin Sulfate ◽  
Biochemical Characterization ◽  
Core Protein ◽  
Chondroitin Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
Protein Length ◽  
Core Proteins ◽  
Leg Muscle

In this article, proteoglycans from embryonic chick leg muscle are quantitatively and qualitatively compared with day 8 high density cell culture cartilage proteoglycans by electron microscopy of proteoglycan-cytochrome c monolayers. The visualized proteoglycan profiles were separated into four categories according to shape, size, and complexity. The two major categories were further characterized by lengths of core proteins, lengths of side projections, and distance between side projections. Two large proteoglycans are identifiable in spread leg muscle preparations. One group has a core protein (mean length of 205 nm) from which extend long thin side projections that we interpret to be groups of chondroitin sulfate glycosaminoglycans with a mean length of 79 nm. This large chondroitin sulfate proteoglycan is the only type found in muscle cultures as determined both biochemically in the past and now by electron microscopy and is referred to as muscle proteoglycan. The second large proteoglycan has a mean core protein length of 250 nm and side projections that are visibly shorter (mean length of 38 nm) and thicker than those of the muscle proteoglycan. This group is referred to as the mesenchymal proteoglycan since its biosynthetic origin is still uncertain. We compare these two profiles with the chick cartilage chondroitin sulfate proteoglycan that has a mean core protein length of 202 nm and side projections with a mean length of 50 nm. The data presented here substantiate the earlier biochemical characterization of these noncartilage proteoglycans and establish the unique structural features of the muscle proteoglycan as compared with the similar profiles of the cartilage and mesenchymal proteoglycans.

scholarly journals Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules.

1993 ◽  
Vol 120 (3) ◽  
pp. 815-824 ◽  
Author(s):  
M Grumet ◽  
A Flaccus ◽  
R U Margolis
Keyword(s):  
Cell Adhesion ◽  
Chondroitin Sulfate ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Core Protein ◽  
Functional Characterization ◽  
Adult Brain ◽  
Chondroitin Sulfate Proteoglycans ◽  
Core Proteins ◽  
Cell Adhesion And Migration

Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system.

scholarly journals Characterization of Vaccinia Virus Intracellular Cores: Implications for Viral Uncoating and Core Structure

2000 ◽  
Vol 74 (8) ◽  
pp. 3525-3536 ◽  
Author(s):  
Ketil Pedersen ◽  
Eric J. Snijder ◽  
Sibylle Schleich ◽  
Norbert Roos ◽  
Gareth Griffiths ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Vaccinia Virus ◽  
Dna Labeling ◽  
The Core ◽  
Double Stranded Dna ◽  
Core Proteins ◽  
Transcription Inhibitor ◽  
Infected Cells ◽  
Combined Data

ABSTRACT The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40–dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.

scholarly journals Chondroitin Sulfate Proteoglycan Expression and Binding of Plasmodium falciparum-Infected Erythrocytes in the Human Placenta during Pregnancy

2003 ◽  
Vol 71 (5) ◽  
pp. 2455-2461 ◽  
Author(s):  
Sean T. Agbor-Enoh ◽  
Rajeshwara N. Achur ◽  
Manojkumar Valiyaveettil ◽  
Rose Leke ◽  
Diane W. Taylor ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Chondroitin Sulfate ◽  
Structural Changes ◽  
Gradual Increase ◽  
First Trimester ◽  
Chondroitin Sulfate Proteoglycans ◽  
Sulfate Content ◽  
Tissue Weight ◽  
Intervillous Space ◽  
Malaria During Pregnancy

ABSTRACT A characteristic feature of malaria during pregnancy is the sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the intervillous spaces of the placenta. We have recently shown that unusually low-sulfated chondroitin sulfate proteoglycans (CSPGs) present in the intervillous spaces mediate the adherence of IRBCs in the placenta. In areas of endemicity, the prevalence of P. falciparum infection in pregnant women peaks during weeks 13 to 20 and then gradually declines, implying that the placental CSPGs are available for IRBC adhesion early during the pregnancy. However, there is no information on the expression and composition of CSPGs during pregnancy. In this study, the expression pattern of CSPGs during the course of pregnancy was investigated. The CSPGs were purified from placentas of various gestational ages, characterized, and tested for the ability to bind IRBCs. The data demonstrate that the CSPGs are present in the intervillous spaces throughout the second and third trimesters. The levels of CSPGs expressed per unit tissue weight were similar in placentas of various gestational ages. However, the structures of the intervillous-space CSPGs changed considerably during the course of pregnancy. In particular, the molecular weight was decreased, with an accompanying gradual increase in the CSPG size polydispersity, from 16 weeks until 38 weeks. The sulfate content was increased considerably after 24 weeks. Despite these structural changes, the CSPGs of placentas of various gestational ages efficiently supported the binding of IRBCs. These results demonstrate that CSPGs can mediate the sequestration of IRBCs in the intervillous spaces of the placenta during the entire second and third trimesters and possibly during the later part of the first trimester as well.

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