Chemical synthesis and receptor binding of catfish somatostatin: a disulfide-bridged beta-D-Galp-(1-->3)-alpha-D-GalpNAc O-glycopeptide.
The glycopeptide hormone catfish somatostatin (somatostatin-22) has the amino acid sequence H-Asp-Asn-Thr-Val-Thr-Ser-Lys-Pro-Leu-Asn-Cys-Met-Asn-Tyr-Phe-Trp-Lys-Se r-Arg-Thr-Ala-Cys-OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains D-GalNAc and D-Gal O-glycosidically linked to Thr5. The linear sequence was assembled smoothly starting with an Fmoc-Cys(Trt)-PAC-PEG-PS support, using stepwise Fmoc solid-phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin-22 were accessed by incorporating as building blocks, respectively, Nalpha-Fmoc-Thr(Ac3-alpha-D-GalNAc)-OH and Nalpha-Fmoc-Thr(Ac4-beta-D-Gal-(1-->3)-Ac2-alpha-D-GalNAc)-O H. Acidolytic deprotection/cleavage of these peptidyl-resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl-protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by Nalpha-dithiasuccinoyl (Dts)-glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2-fold tighter binding.
Chen
L
L
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Jensen
K J
KJ
Tejbrant
J
J
Taylor
J E
JE
Morgan
B A
BA
Barany
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001671:Biochemistry / Q000379:methods; D002397:Catfishes; D004220:Disulfides / Q000737:chemistry; D006023:Glycoproteins / Q000138:chemical synthesis / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D017481:Receptors, Somatostatin / Q000378:metabolism*; D013004:Somatostatin / Q000138:chemical synthesis* / Q000378:metabolism*
D004220:Disulfides; D006023:Glycoproteins; D017481:Receptors, Somatostatin; D013004:Somatostatin