title: Trefoil factor family domains represent highly efficient conformational determinants for N-linked N,N'-di-N-acetyllactosediamine (LacdiNAc) synthesis.
abstract: The disaccharide N,N'-di-N-acetyllactose diamine (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 glycosyltransferases β4GalNAc-T3/T4. Whereas previous studies have revealed evidence for peptide determinants as controlling elements in LacdiNAc biosynthesis, we report here on an entirely independent conformational control of GalNAc transfer by single TFF (Trefoil factor) domains as high stringency determinants. Human TFF2 was recombinantly expressed in HEK-293 cells as a wild type 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 double point-mutated probe (T68Q/F59Q) lacking aromatic residues within a hydrophobic patch. The N-glycosylation probes were analyzed 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 (T68Q/F59Q) 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 extracellular matrix-related LacdiNAc-positive glycoproteins detected as novel candidate proteins in the secretome of HEK-293 cells.
authors.affiliation: From the Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany and.
authors.forename: David
authors.initials: D
authors.lastname: Bonar
authors.affiliation: From the Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany and Center for Molecular Medicine Cologne, University of Cologne, Robert-Koch-Strasse 21, 50931 Köln, Germany franz.hanisch@uni-koeln.de.
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D054794:Glycomics; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007150:Immunohistochemistry; D007785:Lactose / Q000031:analogs & derivatives* / Q000096:biosynthesis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D034421:Sus scrofa; D000071164:Trefoil Factor-2
chemical_list: D006023:Glycoproteins; D050505:Mutant Proteins; D010455:Peptides; D011134:Polysaccharides; D011994:Recombinant Proteins; C000604007:TFF2 protein, human; D000071164:Trefoil Factor-2; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D007785:Lactose
keywords: Carbohydrate Biosynthesis; Glycoprotein; Glycosylation; LDN; LacdiNAc; Mass Spectrometry (MS); N-Linked glycosylation; TFF2; Trefoil Factor Family