Elsevier

Molecular Brain Research

Volume 87, Issue 2, 5 March 2001, Pages 223-237
Molecular Brain Research

Research report
Characterization of mouse brain-specific angiogenesis inhibitor 1 (BAI1) and phytanoyl-CoA alpha-hydroxylase-associated protein 1, a novel BAI1-binding protein

https://doi.org/10.1016/S0169-328X(01)00004-3Get rights and content

Abstract

Previously, PAHX-AP1 (PAHX-associated protein 1) was isolated as a novel protein to interact with Refsum disease gene product (phytanoyl-CoA alpha-hydroxylase, PAHX) and specifically expressed in mouse brain. PAHX-AP1 is also suggested to be involved in the development of the central neurologic deficits of Refsum disease. To clarify its function, we have searched for proteins that associate with PAHX-AP1 via yeast two-hybrid system. We found that PAHX-AP1 interacts with the cytoplasmic region of human brain-specific angiogenesis inhibitor 1 (hBAI1), and isolated murine homolog of hBAI1. Structural analysis of the PAHX-AP1 with three reported hBAI-associated proteins (BAP) revealed no homology among them, and we designated PAHX-AP1 as BAP4. The ability of BAP4 to interact with BAI1 was confirmed by pulling-down BAI1 with GST-BAP4 protein and immunoprecipitation study using brain lysate. Northern and Western blot analyses demonstrated a unique pattern of BAI1 expression in the brain. The peak level of BAI1 was observed 10 days after birth. In situ hybridization analyses of the brain showed the same localization of BAI1 as BAP4, such as most neurons of cerebral cortex, hippocampus, and V, VI, VII, VIII, and XII nuclei. Because BAI1 possessed thrombospondin-type 1 repeats in its extracellular region, changes of BAI1 expression were examined in the focal cerebral ischemia model. The BAI1 expression decreased on the ischemic side after 24 h but BAP4 was not changed after the time-course of ischemia. Our results indicate that expression and localization of BAI1 in the brain is correlated with BAP4, and that BAI1 is involved in inhibition of angiogenesis and neuronal differentiation.

Introduction

We recently isolated a novel brain-specific protein (PAHX-AP1) associated with PAHX, a Refsum disease gene product, using the yeast-based two-hybrid assay [14]. Refsum disease (RfD) is an autosomal recessive disorder of the lipid metabolism. Its major clinical findings include retinitis pigmentosa, peripheral neuropathy, nerve deafness and cerebellar ataxia [32]. We showed a unique targeted expression of PAHX-AP1 in mouse and human brains, and highly conserved amino acid sequences between humans and mice (99%). In situ hybridization analyses of the rat brain showed that PAHX-AP1 is expressed in the most neurons of the cerebral cortex, dentate gyrus, hippocampus, the Purkinje cell layer and deep cerebellar nucleus, trigeminal, abducent, facial, and cochlear nuclei, and retina. It also showed that PAHX-AP1 is mainly expressed in the neuron but not in the oligodendrocyte, which is a major pathologic site of the polyneuropathy in RfD. It indicates that PAHX-AP1 is not involved in the pathogenesis of peripheral polyneuropathy. Interestingly, those subregions of the brain frequently show overt pathologic symptoms in RfD patients, and it is suggested that PAHX-AP1 is involved at least in the development of central neurologic deficits in RfD.

To characterize the function of PAHX-AP1 in the nervous system, we searched for cellular proteins that may associate with it by screening a mouse brain cDNA library with yeast two-hybrid system. Here we report the identification of the interaction between the cytoplasmic region (1254–1516 aa) of BAI1 and the whole encoding region of PAHX-AP1, and cloning of the mouse homolog (mBAI1, 5498 bp) of human BAI1. hBAI1 was reported to have a functional p53 binding sequence, and seven-span transmembrane region as well as extended extracellular and cytoplasmic domains [18]. hBAI1 possessed two presumably functional sites in its extracellular region, an Arg-Gly-Asp (RGD) motif and thrombospondin (TSP)-type 1 repeats. The RGD motif is a potential recognition sequence for binding integrins [4], and the TSP-type 1 repeats can inhibit angiogenesis in the rat cornea induced by bFGF [30]. To date, the functions of BAI1 in the brain are as yet unknown. In this study, we found that BAI1 also acts as an angiogenesis inhibitor in the rat cerebral cortex like the TSP in the cornea.

Three BAI-associated proteins (BAP1, 2, 3) were reported to interact with the cytoplasmic domain of hBAI1 by means of a yeast two-hybrid screening system [20], [22], [24]. However, structural analysis of the PAHX-AP1 revealed that there is no homology between the three BAPs and PAHX-AP1. We renamed PAHX-AP1 as BAP4. Considering the unique expression pattern and the same localization of BAI1 and BAP4 in the brain, interaction of BAI1 with BAP4 might be involved in several important neuronal functions, such as neuronal differentiation and angiogenesis inhibition. It suggests that interaction of PAHX with BAI1 through BAP4 is also related to the pathogenesis of the central neurologic symptoms of RfD.

Section snippets

Yeast two-hybrid assay, isolation and analysis of murine BAI1

The yeast two-hybrid assay was performed as described in the Matchmaker protocol (Clonetech, Palo Alto, CA). Briefly, the whole portion of murine PAHX-AP1 was amplified from the corresponding cDNA by PCR and cloned into the GAL4 DNA-binding domain vector, a bait vector, pGBT9 using the EcoRI and BamHI restriction sites. Y190 yeast cells were co-transformed with the bait vector harboring PAHX-AP1 cDNA and a prey vector containing mouse brain cDNA libraries (Clonetech), then cultured at 30°C in a

Cloning of the murine BAI1 cDNA

To identify new proteins that interact with and potentially regulate PAHX-AP1, a mouse brain cDNA library was screened by the yeast two-hybrid assay using the whole encoding region of murine PAHX-AP1 cDNA as bait. Several positive clones were obtained and data base surveys identified a high degree of the deduced amino acid sequence identity (90%, Fig. 2) between one of the positive clones and hBAI1. This cDNA insert corresponded to the cytoplasmic region (1254–1516 aa) of hBAI1. This murine

Discussion

In this study, we isolated the murine homolog of hBAI1 that interacts with PAHX-AP1, a Refsum disease gene product related protein, using the yeast two-hybrid assay. We revealed a unique expression of mBAI1 in the mouse brain, and highly conserved amino acid sequences between humans and mice (94%). In situ hybridization analyses showed that mBAI1 is co-localized with PAHX-AP1(BAP4) in the rat brain, such as in the most neurons of the cerebral cortex, olfactory bulb, dentate gyrus, hippocampus,

Acknowledgements

We thank S.M. Lee, S.R. Park and K.S. Lee (Chonnam University) for assistance with cloning and cell culture experiments. This study was supported by a grant (HMP-98-N-2-0024) of the 98 Good Health R and D Program, Ministry of Health and Welfare, Republic of Korea.

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