Solid-phase synthesis and evaluation of glycopeptide fragments from rat epididymal cysteine-rich secretory protein-1 (Crisp-1).
Three 18-residue peptides with the sequence Glp-Asp-Thr-Thr-Asp-Glu-Trp-Asp-Arg-Asp-Leu-Glu-Asn-Leu-Ser-Thr-Thr-Lys, taken from the N-terminus of the rat epididymal cysteine-rich secretory protein (Crisp-1) that is important in the fertilization process, were prepared by Fmoc solid-phase synthesis using a convergent strategy. These peptides were the parent sequence, plus two possible α-O-linked T(N) antigen-containing glycopeptides with a Thr(α-D-GalNAc) residue in place of either Thr3 or Thr4. During chain assembly, two deletion peptides [des-Asp2 and des-Thr(Ac(3)-α-D-GalNAc)] and one terminated peptide [N-acetylated 14-mer] arose, as did several peptides in which aspartimide formation had occurred at each of the four possible positions in the sequence. These by-products totaled ~20% of the desired product; they were recognized by HPLC and ESI-MS and removed during the intermediate purifications. Final products, obtained in 15-21% overall yields, were characterized by HPLC purities and ESI-MS. Circular dichroism (CD) spectra for all three purified peptides, recorded in pure water and in trifluoroethanol-H(2)O (1:1), revealed that the presence of a sugar moiety does not significantly impact the sampled conformations. Future biological evaluation could elucidate the nature and locus of sugar modification of Crisp-1, and provide insight as to why Crisp-1 protein E binds sperm irreversibly, in contrast to protein D that lacks a sugar near the N-terminus and only binds sperm loosely.
Liu
Mian
M
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Hamilton
David W
DW
Barany
George
G
D000818:Animals; D030501:Epididymal Secretory Proteins / Q000737:chemistry*; D006020:Glycopeptides / Q000138:chemical synthesis; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000138:chemical synthesis*; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats
C486320:Crisp3 protein, rat; D030501:Epididymal Secretory Proteins; D006020:Glycopeptides; D008562:Membrane Glycoproteins; D010446:Peptide Fragments