Carbon-13 NMR studies of native and modified ovine submaxillary mucin

Biochemistry. 1984 Mar 27;23(7):1485-97. doi: 10.1021/bi00302a023.

Abstract

Natural abundance 13C NMR spectroscopy has been used to study the solution structure and dynamics of the ovine submaxillary mucin (OSM). Results at both 45.3 and 67.9 MHz show the extremely viscous mucin to possess sufficient internal segmental flexibility to allow high-resolution 13C NMR studies. Essentially all of the resonances in the spectra have been assigned to individual carbons of the carbohydrate disaccharide side chain alpha- NeuNAc2 ----6 alpha-Gal-NAc-Ser/Thr and to the protonated carbons of the major peptide residues. Spin-lattice relaxation times and nuclear Overhauser enhancements reveal that the internal mobility of the mucin is unaffected by large changes in molecular weight and hence bulk viscosity. On the basis of the relaxation measurements the peptide and carbohydrate side chain mobilities increase stepwise from the glycosylated peptide residue alpha-carbons to the terminal sialic acid (NeuNAc) side-chain C9 carbon. Removal of the terminal sialic acid C8 and C9 side-chain carbons as well as the complete removal of the NeuNAc residue does not alter the dynamics of the peptide core. However, the removal of carbons C8 and C9 from the NeuNAc residue produces an increase in its ring mobility or conformational flexibility. Complete removal of sialic acid produces an increase in the mobility or flexibility of the GalNAc ring and reduces the chemical shift sensitivity of the GalNAc ring carbons to the different serine and threonine linkages. The pKa value for the sialic acid carboxyl group in the intact mucin is 2.0, while it increases to 2.4 after the removal of the NeuNAc C8 and C9 side-chain carbons. This change in pKa confirms the intramolecular hydrogen bond interaction of the C8 hydroxyl with the C2 carboxyl group in the alpha-NeuNAc residue as previously suggested by Jennings and Bhattacharjee [ Jennings , H.J., & Bhattacharjee , A.K. (1977) Carbohydr . Res. 55, 105-112]. The relaxation time values and temperature dependence of the chemical shift of the NeuNAc C7 carbon suggest that this group is also involved in an intramolecular interaction. Overall the 13C NMR results indicate that the relatively simple mucous glycoprotein, OSM, is a highly extended and internally flexible molecule which in solution possesses little secondary structure.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Glycoproteins
  • Hydrogen-Ion Concentration
  • Magnetic Resonance Spectroscopy
  • Mucins*
  • Protein Conformation
  • Sheep
  • Sialic Acids / analysis
  • Submandibular Gland / analysis
  • Temperature

Substances

  • Glycoproteins
  • Mucins
  • Sialic Acids