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A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line.

ABSTRACT: We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.

SUBMITTER: Cooper S

PROVIDER: S-EPMC1132996 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

Project description:Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

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Cell surface chondroitin sulphate proteoglycan 4 (CSPG4) binds to the basement membrane heparan sulphate proteoglycan, perlecan, and is involved in cell adhesion.

Project description:Chondroitin sulphate proteoglycan 4 (CSPG4) is a cell surface proteoglycan highly expressed by tumour, perivascular and oligodendrocyte cells and known to be involved cell adhesion and migration. This study showed that CSPG4 was present as a proteoglycan on the cell surface of two melanoma cell lines, MM200 and Me1007, as well as shed into the conditioned medium. CSPG4 from the two melanoma cell lines differed in the amount of chondroitin sulphate (CS) decoration, as well as the way the protein core was fragmented. In contrast, the CSPG4 expressed by a colon carcinoma cell line, WiDr, was predominantly as a protein core on the cell surface lacking glycosaminoglycan (GAG) chains. This study demonstrated that CSPG4 immunopurified from the melanoma cell lines formed a complex with perlecan synthesized by the same cultured cells. Mechanistic studies showed that CSPG4 bound to perlecan via hydrophobic protein-protein interactions involving multiple sites on perlecan including the C-terminal region. Furthermore, this study revealed that CSPG4 interacted with perlecan to support cell adhesion and actin polymerization. Together these data suggest a novel mechanism by which CSPG4 expressing cells might attach to perlecan-rich matrices so as those found in connective tissues and basement membranes.

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F9 Embryonal Carcinoma cell line

Project description:Expression profile for undifferentiated F9 Embryonal Carcinoma cell line Keywords: expression profile

2008-02-27 | GSE10606 | GEO

Chondroitin Sulphate Proteoglycan Axonal Coats in the Human Mediodorsal Thalamic Nucleus.

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Keratocan, a cornea-specific keratan sulfate proteoglycan, is regulated by lumican.

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Proteoglycan synthesis in human and murine haematopoietic progenitor cell lines: isolation and characterization of a heparan sulphate proteoglycan as a major proteoglycan from the human haematopoietic cell line TF-1.

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| S-EPMC6647153 | biostudies-literature

Establishment and Characterization of a Novel Human Ocular Adnexal Sebaceous Carcinoma Cell Line.

Project description:PurposeSebaceous carcinoma (SC) is a malignant eyelid tumor of the ocular adnexa that is primarily treated via surgical excision. Few therapies exist in advanced cases, and medical therapy is limited because of our incomplete understanding of SC biology. Herein, we describe a technique to culture human ocular adnexal SC for use as an in vitro model.MethodsHuman ocular adnexal SC tumor cells were isolated from a patient undergoing orbital exenteration surgery and named Bascom Palmer 50 (BP50). They were cultured in Dulbecco's modified Eagle medium/nutrient mixture F-12 supplemented with 10% fetal bovine serum and antibiotics and were maintained at 37°C in humidified 5% CO2. The cells were characterized by immunohistochemistry, exome sequencing, and short tandem repeats analysis. In vitro drug screening against mitomycin-C (MMC) was performed using a cell viability assay.ResultsBP50 grew past 40 passages with a doubling time of 52.3 hours. Immunocytochemical staining revealed expression of SC-associated markers adipophilin, epithelial membrane antigen, p53, and androgen receptor. Whole exome sequencing showed a significant carryover in somatic mutations between the tumor tissue and corresponding cell line, revealing genetic markers consistent with SC. MMC affected cell viability in a dose-dependent manner.ConclusionsBP50 displays characteristics of ocular adnexal SC and therefore may facilitate improved understanding of SC biology and the high throughput assessment of novel therapeutic compounds and new drug combinatorial approaches targeted for this disease.Translational relevanceDrug screening with MMC against these cells shows in vitro evidence to support its continued clinical use in SC.

| S-EPMC8161695 | biostudies-literature

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