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The gene structure and organization of mouse PG-Lb, a small chondroitin/dermatan sulphate proteoglycan.

ABSTRACT: PG-Lb was originally characterized as a small chondroitin/dermatan sulphate proteoglycan expressed preferentially in the zones of flattened chondrocytes in developing chick limb cartilage. The occurrence of this proteoglycan in mammalian cartilage has been shown by the isolation of a cDNA clone from mouse cartilage cDNA library [Kurita, Shinomura,Ujita, Zako, Kida, Iwata and Kimata (1996) Biochem. J. 318, 909-914]. To understand the regulation mechanisms for such a unique expression, we have investigated a genomic DNA structure of the PG-Lb gene. The gene is composed of seven exons and six introns spanning more than 50 kb. The leucine-rich repeats are encoded from exon V to exon VII. The transcription initiation site has been determined by rapid amplification of the cDNA ends ('5'-RACE'). The possible TATA box was detected about 90 bp upstream of the adenosine residue that was numbered as position +1. Further analyses of 1.5 kb of the 5' flanking region and 2.2 kb of the first intron have revealed several potential binding motifs for transcription factors such as Sox 5 and 9. The presence of those sequences in the PG-Lb gene was discussed in relation to the unique expression of this proteoglycan. The chromosomal localization of the murine PG-Lb gene was determined to be on the mouse chromosome 10 by the fluorescence-in-situ-hybridization ('FISH') method.

SUBMITTER: Iwata Y

PROVIDER: S-EPMC1219441 | biostudies-other | 1998 May

REPOSITORIES: biostudies-other

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Occurrence of PG-Lb, a leucine-rich small chondroitin/dermatan sulphate proteoglycan in mammalian epiphyseal cartilage: molecular cloning and sequence analysis of the mouse cDNA.

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Project description:BACKGROUND:Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. METHODS:NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. RESULTS:NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. CONCLUSION:The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

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X-ray-diffraction patterns from chondroitin 4-sulphate, dermatan sulphate and heparan sulphate.

Project description:Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating beta-d-(1-->4) and alpha-d-(1-->4).

| S-EPMC1177740 | biostudies-other

Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta.

Project description:A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200,000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15,000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24,000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.

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