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



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




井上, 明宏


イノウエ, アキヒロ











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


  1. CONTENTS / p2 (0004.jp2)
  2. Abbreviations / p6 (0008.jp2)
  3. Summary / p7 (0009.jp2)
  4. Introduction / p12 (0014.jp2)
  5. I.Cloning,identification and sequence analysis of cDNA for a neuronal cell membrane protein recognized by the monoclonal antibody, HPC-1. / p16 (0018.jp2)
  6. I-1.Materials and Methods / p17 (0019.jp2)
  7. I-2.Results / p23 (0025.jp2)
  8. I-3.Discussion / p31 (0033.jp2)
  9. II.Tissue distribution, cellular and subcellular localization, and developmental expression of HPC-1 immunoreactivity and its mRNA / p39 (0041.jp2)
  10. II-1.Materials and Methods / p40 (0042.jp2)
  11. II-2.Results / p47 (0049.jp2)
  12. II-3.Discussion / p53 (0055.jp2)
  13. Acknowledgments / p61 (0063.jp2)
  14. References / p64 (0066.jp2)


    • 8000000090838
  • DOI(NDL)
  • 本文言語コード
    • eng
  • NDL書誌ID
    • 000000254931
  • データ提供元
    • 機関リポジトリ
    • NDLデジタルコレクション