神経細胞膜抗原ＨＰＣ-１のｃＤＮＡクローニング、一次構造解析及び組織発現様式

井上, 明宏; イノウエ, アキヒロ; INOUE, Akihiro

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神経細胞膜抗原ＨＰＣ-１のｃＤＮＡクローニング、一次構造解析及び組織発現様式

https://ir.soken.ac.jp/records/1071

名前 / ファイル	ライセンス	アクション
要旨・審査要旨 / Abstract, Screening Result (380.3 kB)

Item type

学位論文 / Thesis or Dissertation(1)

公開日

2010-02-22

タイトル

神経細胞膜抗原ＨＰＣ-１のｃＤＮＡクローニング、一次構造解析及び組織発現様式

タイトル

cDNA cloning, sequence analysis and tissue distribution of a neuronal cell membrane antigen, HPC-1

言語

eng

資源タイプ

資源タイプ識別子

http://purl.org/coar/resource_type/c_46ec

資源タイプ

thesis

著者名

井上, 明宏

フリガナ

イノウエ, アキヒロ

著者

INOUE, Akihiro

学位授与機関

学位授与機関名

総合研究大学院大学

学位名

博士（学術）

学位記番号

内容記述タイプ

Other

内容記述

総研大甲第27号

研究科

値

生命科学研究科

専攻

値

20 生理科学専攻

学位授与年月日

1992-03-16

学位授与年度

値

1991

要旨

内容記述タイプ

Other

内容記述

Monoclonal antibody(MAb) is useful tool to study the
molecular basis of development, construction and function of the
nervous system. MAb technique can find unknown molecules
which exhibit characteristic spatial or temporal expression patterns,
possibly playing significant roles on the nervous system.　　A
protein antigen recognized by a MAb, designated HPC-1,localized
in the plasma membrane of the amacrine cell somas and the inner
plexiform layer (IPL) in rat retinae.　　MAb HPC-1 recognized
several proteins of about 35 kDa in SDS-PAGE.
　　　In the chapter I of this thesis, an Mab HPC-1-positive cDNA
clone, HPC-113, was isolated from a rat hippocampus cDNA library
constucted in a lambda phage vector,λgt11, which expressed
β-galactosidase/cDNA fusion protein. The rabbit antiserum raised
against the β-galactosidase/HPC-113 fusion protein showed the
almost same characterisics both in immunoblotting and
immunohistochemical studies as those with MAb HPC-1 in the rat
retina. Thus it was concluded that HPC-113 coded for the antigen
molecule(s) recognized by MAb HPC-1. HPC-113 had 894-bp
nucleotide sequence in the same open reading frame of E. coli
β-galactosidase gene and followed by a 1326-bp possible
3’noncoding sequence, and the calculated molecular weight of the
deduced amino acid (298 residues) was 33989 Da, implying that
HPC-113 contains almost the full-length coding region of HPC-1
mRNA. The hybrophathy profile of the deduced amino acid
sequence showed the presence of an obvious hydrophobic region at the
carboxy-terminal end, suggisting that HPC-1 antigen is an
integranted membrane protein.　　These results were comparable to
the results of biochemical and immunohistochemical studies all of
which indicated that HPC-1 should be tightly associated with
plasma membrane.　　Although HPC-1 antigen sequence had no
typical amino(N)-ternminal signal peptide sequence which was
required for secretory and membrane proteins, it was suggested that
the large N-terminal side was in the extracellular domain, since
MAb HPC-1, of which the epitope was in the N-terminal side,
reacted with living cells.　　Thus, HPC-113 might not include the
complete full-length of the coding region of HPC-1 antigen
molecule. On the other hand, the in vitro tlanscription/translation
product from mithionine, the 11th residue of the 298-deduced
amino acid sequence,co-migrated with the lowest band of HPC-1
antigens detected in the immunoblot analysis.　　The cDNA probe,
the insert of λHPC-113, detected single 2.4-kb mRNA in the RNA
blot analysis, and the S1 nuclase protection analysis probed with
the coding region of cDNA also indicated that there was only single
kind of mRNA for HPC-1 antigen.　　Therefore, it might be possible
that HPC-1 antigen was translated from Met11 and its heterogeneity
was generated by any posttranslational modifications. 　　However,
sufficient understanding about the HPC-1 antigen mRNA,its
primary structure and heterogeneity are not attained at present.
Secondary structure prediction analysis revealed that a part of
HPC-1 antigen protein formed α-herical strcture with the
periodical heptad repeats by hydrophobic amino acids, which was
usually seen in fibrous proteins with dimer or trimer coiled-coil
structures.　　These results implied that HPC-1 molecule might bind
to other proteins by its intra- or inter-polypeptide chain association
capacity.　　Although the whole amino acid sequence did not show
significant homology to any known proteins so far, a few local
sequences in the N-terminal side had notable homologies with some
partial sequences in mouse laminin B1 chain, a subunit of laminin.
Its well known that Iaminin, an extracellular matrix protein showed
various biological function in the nervous system.　　Interestingly
these homologous sequences in laminin B1 were involved in the
fragments which revealed neurite-outgrowth and/or survival
promoting activity.
　　　cDNA clones for bovine HPC-1 antigen were also isolated.
The longest cDNA colne, BHPC-109, revealed high nucleotide
sequence homology in the coding region(91.3%), whereas,
homology of 3’noncoding regions was lower than that of coding
region. The 5’end was similar to that of HPC-113, indicating that
reverse transcriptions were stopped at this 5’ portion of both of rat
and bovine mRNAs of which sequences possessed quite high G-C
contents.　　The comparison between the deduced amino acid
sequences of rat and bovine represented remarkable conservation
(98%), suggesting a physiogical significance of HPC-1 antigen
through the mammalian evolution.
　　In the chapter II, tissue distribution of HPC-1 antigenecity and
its mRNA was studied by biochemical and histochemical methods.
Immunoblot analysis showed that the antiserum against the fusion
protein described above detected several proteins is about 35 kDa in
the neuronal tissues (retina, cerebral contrex, hippocampus,
cerebellum and spianl cord), but no proteins is detected in the non-
neuronal tissues (liver, kidney, heart, muscle and adrenal). 　　On
the immuohistochemistry of rat nervous system, HPC-1 antigen was
also observed specifically in the nervous system: the matrices of
cerebral cortex and hippocampus (Particulaly in stratum radiatum);
molecular layer, membrane of granular cell soma and gromeruli in
cerebellum; gray matter of spinal cord. However, little staining
was detetected in white matters of the central nervous tissues.　　The
RNA blot analysis also indicated nervous system-specific
expression of HPC-1 mRNA. In the nonneuronal tissue, however,
the high sensitive RNA polymerase chain reaction assay revealed
presence small amount of a HPC-1 gene transcript which appeared
to be closely related to but distinguishable from the neuronal HPC-1
gene transcript. In stiu hyblidization was performed by the
nonradioactive ditiction method to identify cellular localization.
HPC-1 mRNA was present in most of neurons in the central and
peripheral nervous systems except for retina. In the retina, signals
were detected in amacrine cells, and also in ganglion cells which
HPC-1 immunoreactivity was not present in the soma, suggesting
selective localization of HPC-1 mRNA in the IPL of the axon
terminal. Amount of HPC-1 antigen(s) gradually increased in
accordance with development or the IPL formation in chick retina.
Considering from accumulation of HPC-1 antigenecity in the
hippocampal stratum radiatum, cerebellar gromeruli,and retinal
IPL, HPC-1 antigen may associate to synaptic formation and/or
maintenance of neurons.
　　　In conclusion, it was proved that HPC-1 antigen(s) was a
novel class of membrane protein(s) of 35 kDa with α-helical
structure containing typical heptad repeats which related to
association between other polypeptide. A few local sequences had
notable homology to some partial laminin sequences that were
included in the fragments baring neurite-outgrowth and/or survival
promoting activity. HPC-1 antigen(s) was expressed predominantly
in the neuronal tissues with characteristic localization, such as
accumulation into synapse-rich regions, but discrepancy between
immunoreactivity and presence of
mRNA in subpopulations of neurons should be solved in future.

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神経細胞膜抗原ＨＰＣ-１のｃＤＮＡクローニング、一次構造解析及び組織発現様式

× 井上, 明宏

× イノウエ, アキヒロ

× INOUE, Akihiro

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