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Structural studies on equine glycoprotein hormones. Amino acid sequence of equine lutropin beta-subunit.
Abstract: The amino acid sequence was determined for equine lutropin beta (eLH beta). Large fragments were derived from reduced, carboxymethylated eLH beta by digestion with Staphylococcus aureus V8 protease, by cyanogen bromide cleavage, and by cleavage of acid-labile Asp-Pro bonds. The fragments were purified by gel filtration and high performance liquid chromatography (HPLC). The fragments were sequenced by automated Edman degradation to establish the primary structure of eLH beta. Some peptides were further digested with chymotrypsin and the resulting peptides purified by HPLC. In addition to sequencing by automated Edman degradation, these were also sequenced by the complementary 5-dimethylaminonaphthalene-1-sulfonyl-Edman procedure which enabled us to directly identify glycosylated amino acids. The eLH beta subunit is a glycoprotein of 149 amino acids containing both N- and O-linked oligosaccharides. It possesses a COOH-terminal extension similar to that seen in human chorionic gonadotropin. Carboxypeptidase Y digestions suggest that the COOH terminus is blocked by glycosylation. Interestingly, the amino acid sequence of eLH beta is identical to that of equine chorionic gonadotropin beta (Sugino, H., Bousfield, G. R., Moore, W. T., and Ward, D. N. (1987)J. Biol. Chem. 262, 8603-8609).
Publication Date: 1987-06-25 PubMed ID: 3298239 The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This study identified the amino acid sequence of equine lutropin beta (eLH beta), a glycoprotein hormone in horses, through various methods including digestion with V8 protease, cyanogen bromide cleavage, and acid-labile Asp-Pro bond cleavage. The study found that eLH beta’s amino acid sequence is identical to that of equine chorionic gonadotropin beta.
Experiment Procedures
- The amino acid sequence of equine lutropin beta (eLH beta), a glycoprotein hormone in horses, was determined via a multi-step process.
- At first, large fragments of eLH beta were obtained via several chemical and enzymatic methods – digestion with Staphylococcus aureus V8 protease, cleavage by cyanogen bromide, and breaking down of acid-labile Asp-Pro bonds.
- The resulting fragments were subsequently purified through processes such as gel filtration and high performance liquid chromatography (HPLC).
- Automated Edman degradation, a sequencing method for proteins, was used to sequence the eLH beta. Some peptides went through further digestion with the enzyme chymotrypsin for additional purification via HPLC.
Key Findings
- Both automated Edman degradation and the complementary 5-dimethylaminonaphthalene-1-sulfonyl-Edman procedure were used to sequence the more purified peptides.
- The researchers were able to identify glycosylated amino acids directly through the aforementioned complementary procedure.
- eLH beta was found to be a glycoprotein composed of 149 amino acids, which consist of both N- and O-linked oligosaccharides.
- Interestingly, the sequence also possesses a COOH-terminal extension that is similar to the one present in human chorionic gonadotropin, another type of hormone.
- This COOH terminus appears to be blocked by glycosylation, as suggested by the results of carboxypeptidase Y digestions.
- The sequence of amino acids in eLH beta was revealed to be identical to that in equine chorionic gonadotropin beta, a significant discovery in the study.
Cite This Article
APA
Bousfield GR, Liu WK, Sugino H, Ward DN.
(1987).
Structural studies on equine glycoprotein hormones. Amino acid sequence of equine lutropin beta-subunit.
J Biol Chem, 262(18), 8610-8620.
Publication
Researcher Affiliations
MeSH Terms
- Amino Acid Sequence
- Animals
- Cyanogen Bromide
- Horses
- Luteinizing Hormone
- Peptide Fragments / analysis
- Peptide Hydrolases
Grant Funding
- AM-09801 / NIADDK NIH HHS
- HD-18210 / NICHD NIH HHS
Citations
This article has been cited 9 times.- Byambaragchaa M, Park SH, Kim SG, Shin MG, Kim SK, Hur SP, Park MH, Kang MH, Min KS. Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells. Curr Issues Mol Biol 2024 Jun 15;46(6):6085-6099.
- Cohen L, Bousfield GR, Ben-Menahem D. The recombinant equine LHβ subunit combines divergent intracellular traits of human LHβ and CGβ subunits. Theriogenology 2015 Jun;83(9):1469-76.
- Antczak DF, de Mestre AM, Wilsher S, Allen WR. The equine endometrial cup reaction: a fetomaternal signal of significance. Annu Rev Anim Biosci 2013 Jan;1:419-42.
- Bernard MP, Lin W, Kholodovych V, Moyle WR. Human lutropin (hLH) and choriogonadotropin (CG) are assembled by different pathways: a model of hLH assembly. J Biol Chem 2014 May 16;289(20):14360-9.
- Pearl CA, Boime I. Sulfation of LH does not affect intracellular trafficking. Mol Cell Endocrinol 2009 Oct 15;309(1-2):76-81.
- Hayashizaki Y, Hiraoka Y, Endo Y, Miyai K, Matsubara K. Thyroid-stimulating hormone (TSH) deficiency caused by a single base substitution in the CAGYC region of the beta-subunit. EMBO J 1989 Aug;8(8):2291-6.
- d'Angelo-Bernard G, Moumni M, Jutisz M, Counis R. Cloning and sequence analysis of the cDNA for the precursor of the beta subunit of ovine luteinizing hormone. Nucleic Acids Res 1990 Apr 25;18(8):2175.
- Campbell RK, Dean-Emig DM, Moyle WR. Conversion of human choriogonadotropin into a follitropin by protein engineering. Proc Natl Acad Sci U S A 1991 Feb 1;88(3):760-4.
- Stockell Hartree A, Renwick AG. Molecular structures of glycoprotein hormones and functions of their carbohydrate components. Biochem J 1992 Nov 1;287 ( Pt 3)(Pt 3):665-79.
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