Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein

The cell envelope (CE) is a vital structure for barrier function in termina lly differentiated dead stratified squamous epithelia, It is assembled by t ransglutaminase (TGase) cross-linking of several proteins, including SPR3 i n certain specialized epithelia normally subjected to mechanical trauma. We have expressed recombinant human SPR3 in order to study its cross-linking properties, It serves as a complete substrate for, and is cross-linked at s imilar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are wide ly expressed in many epithelia, Multiple adjacent glutamines (4, 5, 16, 17, 18, 19 and 167) and lysines (6, 21, 164, 166 and 168) of only head and tai l domain sequences are used for cross-linking. However, each enzyme prefere ntially uses certain residues on the head domain, Moreover, our in vitro da ta suggest a defined temporal order of cross-linking of SPR3 in vivo: It is first cross-linked by TGase 3 into short intra- and inter-chain oligomers which are later further cross-linked to the CE by TGase 1, To investigate t he absence of cross-linking in the central domain (e.g. lysine in position 2 of each of the 16 repeats) we performed structural studies on recombinant SPR3 and on a synthetic peptide containing three repeats of the central do main. 2D H-l NMR spectroscopy, TOCSY and POESY, shows strong and medium int ensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geom etry computation on the basis of intensities of NOEs found generated 50 com patible structures grouped in three main families differing by the number o f H-bonds, These measurements were repeated at different concentrations of trifluoroethanol (TFE) water mixture, an alpha-helical promoting solvent, i n order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not p roduce any variation in the POESY spectrum in the same condition as above, The NMR and circular dichroism data strongly indicate the presence of an or dered (not alpha-helix nor beta-sheet) highly flexible structure in the eig ht amino acids repetitive units of SPR3, confirming the prediction of one p ossible beta-turn per each repeating unit, Thus, biochemical and biophysica l data, strongly support SPR3 to function as a flexible cross-bridging prot ein to provide tensile strength or rigidity to the CE of the stratified squ amous epithelia in which it is expressed.