Nitro-thiocyanobenzoic acid (NTCB) reactivity of cysteines beta100 and beta110 in porcine luteinizing hormone: metastability and hypothetical isomerization of the two disulfide bridges of its beta-subunit seatbelt.
Luteinizing hormone (LH) like all other glycoprotein hormones is composed of two dissimilar subunits, alpha and beta, that are non-covalently associated. The heterodimer is stabilized by a region of the beta-subunit called the "seatbelt" because it wraps around the alpha-subunit and it is fastened by a disulfide bridge between cysteines beta26 and beta110. Although all 22 cysteines of porcine LH (pLH) are engaged in disulfide bridges, we previously showed that the free cysteine-specific reagent NTCB could react with pLH: it slowly cyanylated two cysteines in pLH and there was a close relationship between NTCB reaction with pLH and association/dissociation kinetics of its subunits. Therefore, cysteines beta26 and beta110 were considered as the best candidates for NTCB reaction. In order to identify the NTCB-reactive cysteines in pLH we have performed a mass spectroscopic analysis of the peptides released after mild basic hydrolysis of S-cyanylated pLH and its subunits. Only cysteines beta100 and beta110 were found to react with NTCB. Since these residues are not linked by a disulfide bridge in the crystallographic 3D structure of gonadotropins, it is proposed that their respective counterparts (Cysbeta93 and beta26) do not react with NTCB either because they are shielded from solvent or because they form a transient bridge. In the first hypothesis, both seatbelt bridges would be independently metastable; in the second one, a fast reversible isomerization between bridges beta26-beta110 and beta93-beta100 would occur. Such a reaction could be catalyzed by the previously recognized intrinsic protein disulfide isomerase (PDI) activity of gonadotropins.
Belghazi
Maya
M
INRA-CNRS-Tours University, Service de Spectrométrie de Masse et de Protéomique, Physiologie de la Reproduction et des Comportements, Centre INRA de Tours-Nouzilly, 37380 Nouzilly, France.
Klett
Danièle
D
Cahoreau
Claire
C
Combarnous
Yves
Y
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007986:Luteinizing Hormone / Q000737:chemistry*; D037101:Luteinizing Hormone, beta Subunit / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D013696:Temperature; D013861:Thiocyanates / Q000737:chemistry*
D004220:Disulfides; D037101:Luteinizing Hormone, beta Subunit; D010446:Peptide Fragments; D013861:Thiocyanates; D007986:Luteinizing Hormone; D003545:Cysteine; C008658:2-nitro-5-thiocyanobenzoic acid