Trefoil Factor Family domains represent highly efficient conformational determinants for N-linked N,N’-di-N-acetyllactosediamine (LacdiNAc) synthesis [Glycobiology and Extracellular Matrices]

September 10th, 2014 by Bonar, D., Hanisch, F.-G.

The disaccharide N,N-di-N-acetyllactosediamine (LacdiNAc, GalNAcβ1-4GlcNAcβ) is found in a limited number of extracellular matrix glycoproteins and neuropeptide hormones indicating a protein-specific transfer of GalNAc by the glycosyl transferases β4GalNAc-T3/T4. Whereas previous studies have revealed evidence for peptide determinants as controlling elements in LacdiNAc biosynthesis we here report on an entirely independent conformational control of GalNAc transfer by single TFF domains as high-stringency determinants. Human TFF2 was recombinantly expressed in HEK-293 cells as a wildtype full-length probe (TFF2-Fl: containing TFF domains P1 and P2), as single P1 or P2 domain probes, as a series of Cys/Gly mutant forms with aberrant domain structures, and as a doubly point mutated probe (Trp68Phe59/Gln) lacking aromatic residues within a hydrophobic patch. The N-glycosylation probes were analysed by mass spectrometry for their glycoprofiles. In agreement with natural gastric TFF2 the recombinant full-length and single domain probes expressed nearly exclusively fucosylated LacdiNAc on bi-antennary complex-type chains indicating that a single TFF domain was sufficient to induce transfer of this modification. Contrasting to this, the Cys/Gly mutants showed strongly reduced LacdiNAc levels and instead preponderant LacNAc expression. The probe with point mutations of two highly conserved aromatic residues in loop 3 (Trp68Phe59/Gln) revealed that these are essential determinant components as the probe lacked LacdiNAc expression. The structural features of the LacdiNAc-inducing determinant on human TFF2 are discussed on the basis of crystal structures of porcine TFF2, and a series of ECM-related LacdiNAc-positive glycoproteins detected as novel candidate proteins in the secretome of HEK-293 cells.
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