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 terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including SPR3 in 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 similar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are widely 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 tail domain sequences are used for cross-linking. However, each enzyme preferentially uses certain residues on the head domain. Moreover, our in vitro data 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 the 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 domain. 2D H-1 NMR spectroscopy, TOCSY and ROESY, shows strong and medium intensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geometry computation on the basis of intensities of NOEs found generated 50 compatible structures grouped in three main families differing by the number of H-bonds. These measurements were repeated at different concentrations of trifluoroethanol (TFE)-water mixture, an alpha-helical promoting solvent, in order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not produce any variation in the ROESY spectrum in the same condition as above. The NMR and circular dichroism data strongly indicate the presence of an ordered (not alpha-helix nor beta-sheet) highly flexible structure in the eight amino acids repetitive units of SPR3, confirming the prediction of one possible beta-turn per each repeating unit. Thus, biochemical and biophysical data, strongly support SPR3 to function as a flexible cross-bridging protein to provide tensile strength or rigidity to the CE of the stratified squamous epithelia in which it is expressed.
Steinert
P M
PM
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, MA 20892-2752, USA.
Candi
E
E
Tarcsa
E
E
Marekov
L N
LN
Sette
M
M
Paci
M
M
Ciani
B
B
Guerrieri
P
P
Melino
G
G
D000818:Animals; D002942:Circular Dichroism; D055233:Cornified Envelope Proline-Rich Proteins; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides; D055232:Proline-Rich Protein Domains; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D055233:Cornified Envelope Proline-Rich Proteins; D003432:Cross-Linking Reagents; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C112254:SPRR3 protein, human; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; C105804:transglutaminase 1; D019204:GTP-Binding Proteins