Determination of Disulfide Structure in Agouti-Related Protein (AGRP) by Stepwise Reduction and Alkylation

Abstract

The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.

Human Dickkopf‐1 (huDKK1) protein: Characterization of glycosylation and determination of disulfide linkages in the two cysteine‐rich domains

“…The Asn-linked site is expected to reside at position 225, which is the only consensus Asn-linked sequence (N-S-S) in huDKK1. The lower molecular weight form of huDKK1 (27,402 Da) is 649 Da heavier than the predicted mature nonglycosylated form of huDKK1 (26,753 Da), suggesting the presence of a potential O-linked glycan. The molecular weight of DKK1 reported in earlier studies appeared to be higher than the data presented here.…”

“…Ser 27 and Ser 30 of N 13 LPPPLGGAAGHPGSAV-SAAPGILYPGGNK 42 are both candidates to be the site of O-linked sugar attachment. Unsuccessful attempts were made using both CID and ETD fragmentation of peptides D and E to assign the glycosylation site (data not shown).…”

α-Melanocyte-Stimulating Hormone Is Contained in Nerve Terminals Innervating Thyrotropin-Releasing Hormone-Synthesizing Neurons in the Hypothalamic Paraventricular Nucleus and Prevents Fasting-Induced Suppression of Prothyrotropin-Releasing Hormone Gene Expression

“…These observations raise the possibilities that AGRP may have actions on pro-TRH neurons independent of its antagonism of ␣-MSH, either directly on the melanocortin receptor itself or through a separate receptor (Graham et al, 1997;Bures et al, 1998), and that only a subpopulation of hypophysiotropic pro-TRH neurons in the PVN is under direct control by ␣-MSH. Nevertheless, whereas fasted animals receiving only vehicle intracerebroventricularly showed a significant reduction in pro-TRH mRNA in the PVN compared with fed controls, as reported previously (Blake et al, 1991;Legradi et al, 1997), the administration of 300 ng of ␣-MSH intracerebroventricularly every 6 hr completely reversed the effect of fasting on pro-TRH gene expression, resulting in a hybridization pattern that was identical to that of the fed animals in all parvocellular subdivisions of the PVN.…”

High-Resolution NMR Structure of the Chemically-Synthesized Melanocortin Receptor Binding Domain AGRP(87−132) of the Agouti-Related Protein,

“…Identical disulfide maps have been determined for both the 112-residue protein and AGRP(85-132) (12,14). A detailed solution structure of the AGRP C-terminal domain is essential for interpretation of AGRP/MCR interactions and for understanding AGRP's unique receptor subtype specificity for MC3R and MC4R.…”