Elsevier

Molecular Brain Research

Volume 77, Issue 1, 14 April 2000, Pages 19-28
Molecular Brain Research

Research report
The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain

https://doi.org/10.1016/S0169-328X(00)00036-XGet rights and content

Abstract

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10.5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Introduction

The transient expression of an adhesion molecule is a crucial event for neural network formation in the early developmental stages of the brain when neurons are most actively engaged in cell–cell interactions. Cell surface adhesion molecules are thought to play a crucial role in axon guidance and fasciculation in the developing nervous system. Membrane proteins on nerve growth cones and growing axons act as recognition molecules involved in neurite extension, pathfinding and targeting of appropriate cells, and consequently the formation of neural connections [7], [11].

We purified membrane proteins exhibiting developmental changes, and determined the partial amino acid sequences of more than 130 peptides obtained on proteolytic cleavage of the membrane proteins [1]. Among them we focused on a membrane protein with a molecular mass of 46 kDa, which was found to be expressed extensively in prenatal brains but to be decreased in adult brains on Western blot analysis with antibodies against the purified peptides. We cloned cDNAs by RT-PCR with degenerate oligonucleotides corresponding to the sequences of the purified peptides and found that this membrane protein consists of two immunoglobulin domains, one V-like and one C2-like domain, followed by a transmembrane domain and a cytoplasmic domain. This structure is found in a number of proteins in the immune system [25] rather than in proteins expressed in the central nervous system.

In the course of our study, Bergelson et al. [2] and Tomko et al. [22] isolated a coxsackievirus and adenovirus receptor (CAR) cDNA. The 46-kDa membrane protein purified from newborn mouse brain was identified as the mouse homologue (mCAR) [2], [3] of the human CAR on prediction of the amino acid sequence from the cDNA. Coxsackievirus is known to exhibit affinity for newborn tissues [6], [13], and was first isolated on intracerebral inoculation into newborn mice. Suckling mice, 3–7 days of age, became paralyzed, while mice of 10–12 g in weight did not [6]. This critical feature of coxsackievirus pathogenicity coincided with our observation of abundant expression up to birth followed by sharp decreases in both the protein and mRNA, and no detection in adult brains. However, the endogenous native function of mCAR during the development of the mouse brain has not been elucidated.

We report here the structural features of mCAR, a member of the immunoglobulin superfamily in the brain, and show its subcellular localization in cultured hippocampal neurons, and the developmental changes in the levels of mCAR and its mRNA. We further demonstrate the cell adhesion activity of mCAR toward cultured cells transfected with cDNA in a eukaryotic expression vector.

Section snippets

Purification of membrane proteins

Growth cone-enriched and non-enriched fractions were prepared from newborn mouse brains by the discontinuous sucrose density gradient centrifugation method [18]. Membrane proteins were isolated from each fraction and digested with lysylendopeptidase, and then the purified proteins were subjected to amino acid microsequencing [1].

Western blot analysis

Two peptides (a, KIYDNYYPDLKC; and b, KTQYNQVPSEDFERAPQC) were synthesized and used to immunize white rabbits as described previously [1]. Mouse brains were homogenized

Isolation of a membrane protein and cDNA cloning

A membrane protein exhibiting a molecular mass of 46 kDa on SDS–PAGE was purified from the growth cone-enriched fraction. To determine whether peptide-a and -b were generated from a single protein, RT-PCR analysis were carried out with two different combinations of oligonucleotide primers, aF and bR, and bF and aR. The primer combination of sense strand aF for peptide-a and anti-sense strand bR for peptide-b produced a 729-bp long DNA fragment. But the other set of primers, bF and aR, failed to

Discussion

We describe here the characterization of a membrane protein purified from a growth cone-enriched fraction which is identical with mCAR [2], [3]. As judged on amino acid sequence comparison, the mCAR is a member of the immunoglobulin superfamily, of which the highest identity of the primary structure is with CTX [5] and human A33-antigen [9]. CTX and A33-antigens are composed of a combination of one V- and one C-like immunoglobulin domain, a hydrophobic transmembrane domain and an intracellular

Acknowledgements

We thank Drs Tadahiro Hamada and Akiko Nishiyama for the helpful discussions, and Dr Hiroaki Asou for the valuable comments on the aggregation assay. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture, and the Ministry of Health and Welfare of Japan (to RK).

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    1

    These authors contributed equally to this work.

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