Substrate Requirements for Transglutaminases

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123

110

␣-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of ␣-crystallin for aggregationprone proteins is probably based on hydrophobic interactions. ␣-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that ␣-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five ␣A-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of ␣A-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.The composition of the vertebrate eye lens is dominated by a group of structural proteins known as crystallins. The largest of these, ␣-crystallin, is a dynamic multimeric complex composed of two types of homologous subunits, ␣A-and ␣B-crystallin (1). A few years ago, it was discovered that these subunits are also constitutively expressed in various non-lenticular tissues, suggesting that their function is more than merely structural (2-5). In addition, increased expression of ␣B-crystallin has been observed in a variety of neurodegenerative disorders such as multiple sclerosis (6), Alexander's disease (7,8), and Alzheimer's disease (9, 10). Although the physiological significance of this extralenticular expression is unknown, it certainly relates to the fact that ␣-crystallins belong to the family of small heat shock proteins (hsp) 1 (11, 12). Among the shared features of ␣-crystallins and other small hsp are the conferring of thermotolerance (13,14), interaction with actin (15, 16), phosphorylation (17, 18), and intracellular relocalization upon stress (19,20). Furthermore, in vitro experiments have shown that ␣A-and ␣B-crystallin as well as hsp25 can act as molecular chaperones by suppressing aggregation of denaturing proteins (21-23). Unfortunately, the mechanism of this functional activity is unclear because the three-dimensional structure of ␣-crystallin is unknown.Native ␣-crystallins occur as polydisperse particles with an average molecular mass of about 800 kDa. Understanding the multimeric arra...

Section: Resultsmentioning

confidence: 99%

Immobilization of the C-terminal Extension of Bovine αA-Crystallin Reduces Chaperone-like Activity

Smulders

Carver

Lindner

et al. 1996

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123

110

“…It is thought that transglutaminase enzymes show greater specificity for glutamines than for lysines (2,32), and this is certainly the case for cross-linking of PAI-2, where only glutamines 83 and 86 are active in cross-linking. The factors that determine whether a glutamine or a lysine can be cross-linked are unclear, but structure of the surrounding sequence is important (33,34). Accessibility on a solvent-exposed region may also play a role (33), as is the case for glutamines 83 and 86 of PAI-2, which are located on an exposed loop.…”

Section: Discussionmentioning

confidence: 99%

Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)

Ritchie¹,

Lawrie²,

Crombie³

et al. 2000

In this study, we identified lysine residues in the fibrinogen A␣ chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with ␣ 2 -antiplasmin (␣ 2 -AP), which is known to cross-link to lysine 303 of the A␣ chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to ␣ 2 -AP but not for crosslinking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-␣C fibrinogens, which lack 100 and 390 amino acids from the C terminus of the A␣ chain, respectively. PAI-2 or ␣ 2 -AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the A␣ chain. ␣ 2 -AP was crosslinked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with ␣ 2 -AP, and the two proteins utilized different lysines in the A␣ chain. Therefore, PAI-2 and ␣ 2 -AP can cross-link simultaneously to the ␣ polymers of a fibrin clot and promote resistance to lysis.

“…Biotinylated Peptides-Six hexapeptides with an NH 2 -terminal biotin followed by a C 6 -spacer were synthesized: GQDPVK, GQDPVR, GNDPVK, GNDPVR, KVPDQG and GKDPVQ (Eurosequence Inc., Groningen, The Netherlands). 250 g of a hexapeptide (TVQQEL) and a heptapeptide (PGGQQIV), two known acyl donor probes in the TGase assay (29), were biotinylated with 500 g of NHS-LC-biotin (Pierce) for 2 h at 37°C in 300 l of 0.1 M Na 2 CO 3 , pH 8.0. The reaction was stopped by the addition of 20 l of 1 M Tris, pH 8.0 (30).…”

Section: Methodsmentioning

confidence: 99%

“…Family-To characterize further the biochemical properties of the above mentioned TGase consensus substrate motif, the biotinylated hexapeptide GQDPVK was synthesized, and enzyme kinetic experiments were performed using a TGase assay (29) to determine optimal reaction conditions. The TGase cross-linking reaction is based on a Ca 2ϩ -dependent exchange of primary amines for ammonia at the ␥-carboxamide group of glutamine residues.…”

Section: Conservation Of a Tgase Substrate Motif In Members Of The Trmentioning

confidence: 99%

Identification and Sequence Analysis of Two New Members of the SKALP/elafin and SPAI-2 Gene Family

Zeeuwen¹,

Hendriks²,

Jong³

et al. 1997

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The human epithelial proteinase inhibitor SKALP/elafin and the porcine sodium-potassium ATPase inhibitor SPAI-2 are two highly homologous proteins that share an NH 2 -terminal transglutaminase substrate domain and a COOH-terminal whey acidic protein (WAP) domain. Here we describe the bovine and simian orthologs of SKALP/elafin as well as two new bovine family members that are designated Trappin-4 and Trappin-5 on the basis of a new nomenclature that we propose (Trappin ‫؍‬ TRansglutaminase substrate and WAP motif-containing ProteIN). Sequence analysis of Trappin-4 and Trappin-5 revealed a domain structure that is very similar to SPAI-2 (Trappin-1) and SKALP/elafin (Trappin-2). The transglutaminase substrate motifs are conserved although the number of repeats varies among species and among family members. The sequence of Trappin-4 and Trappin-5 diverges from Trappin-1 and Trappin-2 at the putative reactive site in the WAP domain. The bovine ortholog of Trappin-2 is expressed in tongue and snout epidermis; Trappin-4 is expressed in trachea, ileum, and tongue; and Trappin-5 is expressed at low levels in trachea, as determined by RNase protection and Northern blot analysis. Based on the analysis of 67 transglutaminase substrate repeats as present in all known Trappin gene family members from four different mammalian species a consensus sequence could be established: GlyGln-Asp-Pro-Val-Lys (GQDPVK). Using biotinylated hexapeptide probes we found that the GQDPVK sequence is a very efficient transglutaminase substrate both for guinea pig liver transglutaminase and for epidermal transglutaminase, and it acts as acyl donor as well as acceptor. We propose that the Trappin protein family forms a new group of enzyme inhibitors with various specificities of the WAP domain, which share transglutaminase substrate motifs that can act as an anchoring sequence. Skin-derived antileukoproteinase (SKALP)1 (1) and sodiumpotassium ATPase inhibitor-2 (SPAI-2) (2) are two molecules that share an NH 2 -terminal domain that functions as a transglutaminase (TGase) substrate, and a COOH-terminal whey acidic protein (WAP) domain that harbors an inhibitory activity toward at least two distinct enzymes. Porcine SPAI-2 was the first of these molecules to be described and is expressed mainly in the intestine (3). Human SKALP, otherwise known as elafin (4), or elastase-specific inhibitor (5), is a potent inhibitor of the leukocytic proteinases elastase and proteinase-3 (6, 7). We found that SKALP/elafin is expressed in several human stratifying squamous epithelia, except for epidermis where it is only expressed in the context of inflammation, such as psoriasis or wound healing (8 -10). We mapped the genomic localization of human SKALP/elafin to chromosome 20q12-13 (11). Interestingly, this region contains various other genes involved in TGase-mediated cross-linking processes such as the tissue TGase gene (12), epidermal TGase (13, 14), and the genes coding for semenogelin I and semenogelin II (15), which are also epithelial TGase substrates. SKAL...