Identification of the crustacean hyperglycemic hormone (CHH) and CHH-like peptides in the crayfish Procambarus clarkii and localization of functionally important regions of the CHH

Hsin Ju Wu, Wei Shiun Tsai, Shao Yen Huang, Yan Jhou Chen, Ying Hsin Chen, Yu Ru Hsieh, Chi Ying Lee

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Four anti-peptide antibodies were raised each against a synthetic peptide corresponding in sequence to a short stretch of the crustacean hyperglycemic hormone (CHH) or CHH-like (CHH-L) peptides of the crayfish Procambarus clarkii. CHH and CHH-L are alternatively spliced products that share an identical sequence for the 1st 40 residues from the amino-terminus of the peptides. When used in Western blot analyses of tissue proteins, anti-CHH (1-10) recognized an immunoreactive protein band in both sinus gland (SGs) and thoracic ganglia (TGs), whereas anti-D-CHH (1-10) recognized an immunoreactive protein band only in SGs, but not in TGs; anti-CHH (59-72) recognized an immunoreactive protein band in SGs but not in TGs, and conversely, anti-CHH-L (58-72) recognized an immunoreactive protein band in TGs but not in SGs. Tissue homogenates were fractionated using high-performance liquid chromatography (HPLC). The immunoreactivity of the collected HPLC fractions was determined by an enzyme-linked immunosorbent assay and Western blotting, and the immunoreactive fractions were subjected to mass determination. A pair of stereoisomers, CHH and D-Phe3 CHH, both with a mass of 8386.4 and respectively immunoreactive to anti-CHH (1-10) and anti-DCHH (1-10), was identified in SGs; Western blot analyses showed that they were immunoreactive to anti-CHH (59-72), but not to anti-CHH-L (58-72). A CHH-L, with a mass of 8343.6 and immunoreactive to anti-CHH (1-10) but not to anti-D-CHH (1-10), was identified in TGs; Western blot analyses showed that it was immunoreactive to anti-CHH-L (58-72), but not to anti-CHH (59-72), and sequencing analysis of the peptide fragments generated by enzyme digestion of the immunoreactive protein revealed 3 sequences, which are contained within a CHH-L encoded by a previously identified transcript. Furthermore, anti-peptide antibodies were tested for the effects of blocking CHH-induced hyperglycemia. Results showed that anti-CHH (59-72) and anti-CHH (1-10) individually abolished CHH-induced hyperglycemia, whereas neither control treatments, pre-immune sera, nor anti-CHH-L (58-72) significantly affected CHH-induced hyperglycemia. In summary, these data reiterate the observations that CHH and CHH-L are preferentially expressed in different tissues; they also suggest that enzymes involved in L-to-D isomerization of CHH are expressed in tissue-specific manners. Finally, the data suggest the N- and C-terminal regions of CHH are important for its biological activity.

Original languageEnglish
Pages (from-to)288-297
Number of pages10
JournalZoological Studies
Volume51
Issue number3
Publication statusPublished - 2012 May 1

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Procambarus clarkii
crayfish
Crustacea
hormones
peptides
thoracic ganglia
sinuses
Western blotting
hyperglycemia
proteins

All Science Journal Classification (ASJC) codes

  • Animal Science and Zoology

Cite this

Wu, Hsin Ju ; Tsai, Wei Shiun ; Huang, Shao Yen ; Chen, Yan Jhou ; Chen, Ying Hsin ; Hsieh, Yu Ru ; Lee, Chi Ying. / Identification of the crustacean hyperglycemic hormone (CHH) and CHH-like peptides in the crayfish Procambarus clarkii and localization of functionally important regions of the CHH. In: Zoological Studies. 2012 ; Vol. 51, No. 3. pp. 288-297.
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title = "Identification of the crustacean hyperglycemic hormone (CHH) and CHH-like peptides in the crayfish Procambarus clarkii and localization of functionally important regions of the CHH",
abstract = "Four anti-peptide antibodies were raised each against a synthetic peptide corresponding in sequence to a short stretch of the crustacean hyperglycemic hormone (CHH) or CHH-like (CHH-L) peptides of the crayfish Procambarus clarkii. CHH and CHH-L are alternatively spliced products that share an identical sequence for the 1st 40 residues from the amino-terminus of the peptides. When used in Western blot analyses of tissue proteins, anti-CHH (1-10) recognized an immunoreactive protein band in both sinus gland (SGs) and thoracic ganglia (TGs), whereas anti-D-CHH (1-10) recognized an immunoreactive protein band only in SGs, but not in TGs; anti-CHH (59-72) recognized an immunoreactive protein band in SGs but not in TGs, and conversely, anti-CHH-L (58-72) recognized an immunoreactive protein band in TGs but not in SGs. Tissue homogenates were fractionated using high-performance liquid chromatography (HPLC). The immunoreactivity of the collected HPLC fractions was determined by an enzyme-linked immunosorbent assay and Western blotting, and the immunoreactive fractions were subjected to mass determination. A pair of stereoisomers, CHH and D-Phe3 CHH, both with a mass of 8386.4 and respectively immunoreactive to anti-CHH (1-10) and anti-DCHH (1-10), was identified in SGs; Western blot analyses showed that they were immunoreactive to anti-CHH (59-72), but not to anti-CHH-L (58-72). A CHH-L, with a mass of 8343.6 and immunoreactive to anti-CHH (1-10) but not to anti-D-CHH (1-10), was identified in TGs; Western blot analyses showed that it was immunoreactive to anti-CHH-L (58-72), but not to anti-CHH (59-72), and sequencing analysis of the peptide fragments generated by enzyme digestion of the immunoreactive protein revealed 3 sequences, which are contained within a CHH-L encoded by a previously identified transcript. Furthermore, anti-peptide antibodies were tested for the effects of blocking CHH-induced hyperglycemia. Results showed that anti-CHH (59-72) and anti-CHH (1-10) individually abolished CHH-induced hyperglycemia, whereas neither control treatments, pre-immune sera, nor anti-CHH-L (58-72) significantly affected CHH-induced hyperglycemia. In summary, these data reiterate the observations that CHH and CHH-L are preferentially expressed in different tissues; they also suggest that enzymes involved in L-to-D isomerization of CHH are expressed in tissue-specific manners. Finally, the data suggest the N- and C-terminal regions of CHH are important for its biological activity.",
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Identification of the crustacean hyperglycemic hormone (CHH) and CHH-like peptides in the crayfish Procambarus clarkii and localization of functionally important regions of the CHH. / Wu, Hsin Ju; Tsai, Wei Shiun; Huang, Shao Yen; Chen, Yan Jhou; Chen, Ying Hsin; Hsieh, Yu Ru; Lee, Chi Ying.

In: Zoological Studies, Vol. 51, No. 3, 01.05.2012, p. 288-297.

Research output: Contribution to journalArticle

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T1 - Identification of the crustacean hyperglycemic hormone (CHH) and CHH-like peptides in the crayfish Procambarus clarkii and localization of functionally important regions of the CHH

AU - Wu, Hsin Ju

AU - Tsai, Wei Shiun

AU - Huang, Shao Yen

AU - Chen, Yan Jhou

AU - Chen, Ying Hsin

AU - Hsieh, Yu Ru

AU - Lee, Chi Ying

PY - 2012/5/1

Y1 - 2012/5/1

N2 - Four anti-peptide antibodies were raised each against a synthetic peptide corresponding in sequence to a short stretch of the crustacean hyperglycemic hormone (CHH) or CHH-like (CHH-L) peptides of the crayfish Procambarus clarkii. CHH and CHH-L are alternatively spliced products that share an identical sequence for the 1st 40 residues from the amino-terminus of the peptides. When used in Western blot analyses of tissue proteins, anti-CHH (1-10) recognized an immunoreactive protein band in both sinus gland (SGs) and thoracic ganglia (TGs), whereas anti-D-CHH (1-10) recognized an immunoreactive protein band only in SGs, but not in TGs; anti-CHH (59-72) recognized an immunoreactive protein band in SGs but not in TGs, and conversely, anti-CHH-L (58-72) recognized an immunoreactive protein band in TGs but not in SGs. Tissue homogenates were fractionated using high-performance liquid chromatography (HPLC). The immunoreactivity of the collected HPLC fractions was determined by an enzyme-linked immunosorbent assay and Western blotting, and the immunoreactive fractions were subjected to mass determination. A pair of stereoisomers, CHH and D-Phe3 CHH, both with a mass of 8386.4 and respectively immunoreactive to anti-CHH (1-10) and anti-DCHH (1-10), was identified in SGs; Western blot analyses showed that they were immunoreactive to anti-CHH (59-72), but not to anti-CHH-L (58-72). A CHH-L, with a mass of 8343.6 and immunoreactive to anti-CHH (1-10) but not to anti-D-CHH (1-10), was identified in TGs; Western blot analyses showed that it was immunoreactive to anti-CHH-L (58-72), but not to anti-CHH (59-72), and sequencing analysis of the peptide fragments generated by enzyme digestion of the immunoreactive protein revealed 3 sequences, which are contained within a CHH-L encoded by a previously identified transcript. Furthermore, anti-peptide antibodies were tested for the effects of blocking CHH-induced hyperglycemia. Results showed that anti-CHH (59-72) and anti-CHH (1-10) individually abolished CHH-induced hyperglycemia, whereas neither control treatments, pre-immune sera, nor anti-CHH-L (58-72) significantly affected CHH-induced hyperglycemia. In summary, these data reiterate the observations that CHH and CHH-L are preferentially expressed in different tissues; they also suggest that enzymes involved in L-to-D isomerization of CHH are expressed in tissue-specific manners. Finally, the data suggest the N- and C-terminal regions of CHH are important for its biological activity.

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