Genomic Organization and Expression Pattern of Mouse Neuroglycan C in the Cerebellar Development

Sachiko Aono,Hiroomi Keino,Takao Ono,Yoko Yasuda,Yoshihito Tokita,Fumiko Matsui,Masahiko Taniguchi,Shin-Ichi Sonta,Atsuhiko Oohira

doi:10.1074/jbc.275.1.337

Abstract

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan with an epidermal growth factor module that is expressed predominantly in the brain. Cloning studies with mouse NGC cDNA revealed the expression of three distinct isoforms (NGC-I, -II, and -III) in the brain and revealed that the major isoform showed 94. 3% homology with the rat counterpart. The NGC gene comprised six exons, was approximately 17 kilobases in size, and was assigned to mouse chromosome band 9F1 by fluorescence in situ hybridization. Western blot analysis demonstrated that, although NGC in the immature cerebellum existed in a proteoglycan form, most NGC in the mature cerebellum did not bear chondroitin sulfate chain(s), indicating that NGC is a typical part-time proteoglycan. Immunohistochemical studies showed that only the Purkinje cells were immunopositive in the cerebellum. In the immature Purkinje cells, NGC, probably the proteoglycan form, was immunolocalized to the soma and thick dendrites on which the climbing fibers formed synapses, not to the thin branches on which the parallel fibers formed synapses. This finding suggests the involvement of NGC in the differential adhesion and synaptogenesis of the climbing and parallel fibers with the Purkinje cell dendrites.

Highlights

Proteoglycans are a group of proteins that bear covalently bound sulfated glycosaminoglycan chains
Cloning of the Mouse neuroglycan C (NGC) cDNA—When a cDNA library (500,000 clones) of the mouse brain was screened for the NGCspecific sequence using a rat cDNA [13] as a probe, more than 400 colonies gave a positive signal above background
The isolated plasmids had a cDNA insert ranging from 2.1 to 2.6 kilobases in size. Most of these cDNA inserts covered the full length of the coding region predicted from the sequence of the rat NGC cDNA

Summary

EXPERIMENTAL PROCEDURES

CDNA Cloning of Mouse NGC—NGC phage clones were isolated from a ␭ ZAP cDNA library derived from the brain of a 2-month-old C57BL/6 strain mouse using a rat NGC cDNA [13] as a probe. Positive plaques were purified by two additional rounds of screening and the excision of the pBluescript phagemid from the ZAP vector (Stratagene, La Jolla, CA) was carried out. Both complementary strands of the mouse NGC cDNA were sequenced using BcaBEST Dideoxy Sequencing kit, dCTP version (TaKaRa, Osaka, Japan). Restriction fragments that included all exon sequences and intron/ exon splice junctions were subcloned into pBluescript phagemid (Stratagene) for DNA sequence analysis.

NGC in the Mouse Cerebellum

RESULTS

TCAGGgtaac tccagGACGA O

When the parts of the NGC cDNAs corresponding to exons

DISCUSSION