PMID- 10510474
OWN - NLM
STAT- MEDLINE
DCOM- 20000104
LR  - 20081121
IS  - 1350-9047 (Print)
IS  - 1350-9047 (Linking)
VI  - 6
IP  - 9
DP  - 1999 Sep
TI  - Transglutaminase crosslinking and structural studies of the human small proline
      rich 3 protein.
PG  - 916-30
AB  - 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.
FAU - Steinert, P M
AU  - Steinert PM
AD  - Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal
      and Skin Diseases, National Institutes of Health, Bethesda, Maryland, MA
      20892-2752, USA.
FAU - Candi, E
AU  - Candi E
FAU - Tarcsa, E
AU  - Tarcsa E
FAU - Marekov, L N
AU  - Marekov LN
FAU - Sette, M
AU  - Sette M
FAU - Paci, M
AU  - Paci M
FAU - Ciani, B
AU  - Ciani B
FAU - Guerrieri, P
AU  - Guerrieri P
FAU - Melino, G
AU  - Melino G
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Cell Death Differ
JT  - Cell death and differentiation
JID - 9437445
RN  - 0 (Cornified Envelope Proline-Rich Proteins)
RN  - 0 (Cross-Linking Reagents)
RN  - 0 (Peptides)
RN  - 0 (Proteins)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (SPRR3 protein, human)
RN  - EC 2.3.2.- (transglutaminase 2)
RN  - EC 2.3.2.13 (Transglutaminases)
RN  - EC 2.3.2.13 (transglutaminase 1)
RN  - EC 3.6.1.- (GTP-Binding Proteins)
SB  - IM
MH  - Animals
MH  - Circular Dichroism
MH  - Cornified Envelope Proline-Rich Proteins
MH  - Cross-Linking Reagents
MH  - GTP-Binding Proteins/metabolism
MH  - Gene Expression
MH  - Humans
MH  - Kinetics
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - Nuclear Magnetic Resonance, Biomolecular
MH  - *Peptides
MH  - Proline-Rich Protein Domains
MH  - Protein Structure, Secondary
MH  - Proteins/*chemistry/genetics/isolation & purification/*metabolism
MH  - Recombinant Fusion Proteins/chemistry/genetics/isolation &
      purification/metabolism
MH  - Sequence Analysis
MH  - Substrate Specificity
MH  - Transglutaminases/*metabolism
EDAT- 1999/10/08 00:00
MHDA- 1999/10/08 00:01
CRDT- 1999/10/08 00:00
PHST- 1999/10/08 00:00 [pubmed]
PHST- 1999/10/08 00:01 [medline]
PHST- 1999/10/08 00:00 [entrez]
AID - 10.1038/sj.cdd.4400568 [doi]
PST - ppublish
SO  - Cell Death Differ. 1999 Sep;6(9):916-30. doi: 10.1038/sj.cdd.4400568.