Zuzana Valnickova

Purification and Characterization of Mouse Soluble Receptor for Advanced Glycation End Products (sRAGE)

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface proteins that has been implicated as a progression factor in a number of pathologic conditions from chronic inflammation to cancer to Alzheimers disease. In such conditions, RAGE acts to facilitate pathogenic processes. Its secreted isoform, soluble RAGE or sRAGE, has the ability to prevent RAGE signaling by acting as a decoy. sRAGE has been used successfully in animal models of a range of diseases to antagonize RAGE-mediated pathologic processes. In humans, sRAGE results from alternative splicing of RAGE mRNA. This study was aimed to determine whether the same holds true for mouse sRAGE and, in addition, to biochemically characterize mouse sRAGE. The biochemical characteristics examined include glycosylation and disulfide patterns. In addition, sRAGE was found to bind heparin, which may mediate its distribution in the extracellular matrix and cell surfaces of tissues. Finally, our data indicated that sRAGE in the mouse is likely produced by carboxyl-terminal truncation, in contrast to the alternative splicing mechanism reported in humans.

Pigment-epithelium-derived factor (PEDF) occurs at a physiologically relevant concentration in human blood: purification and characterization.

Pigment epithelium-derived factor (PEDF) inhibits the formation of blood vessels in the eye by inducing apotosis in actively dividing endothelial cells. The activity of PEDF equals or supersedes that of other anti-angiogenic factors, including angiostatin, endostatin and thrombospondin-1. In addition, PEDF has the potential to promote the survival of neurons and affect their differentiation. Here we show that PEDF is present in plasma at a concentration of approx. 100 nM (5 microg/ml) or twice the level required to inhibit aberrant blood-vessel growth in the eye. Thus the systemic delivery of PEDF has the potential to affect angiogenesis or neurotrophic processes throughout the body, significantly expanding the putative physiological role of the protein. A complete map of all post-translational modifications revealed that authentic plasma PEDF carries an N-terminal pyroglutamate blocking group and an N-linked glycan at position Asn266. The pyroglutamate residue may regulate the activity of PEDF analogously to the manner in which it regulates thyrotropin-releasing hormone.

The Heparin-binding Domain of Extracellular Superoxide Dismutase Is Proteolytically Processed Intracellularly during Biosynthesis

Extracellular superoxide dismutase (EC-SOD) is the only known extracellular enzyme designed to scavenge the superoxide anion. The purified enzyme exists in two forms when visualized by reduced SDS-polyacrylamide gel electrophoresis: (i) intact EC-SOD (Trp1–Ala222) containing the C-terminal heparin-binding domain and (ii) cleaved EC-SOD (Trp1–Glu209) without the C-terminal heparin-binding domain. The proteolytic event(s) leading to proteolysis at Glu209–Arg210 and removal of the heparin-binding domain are not known, but may represent an important regulatory mechanism. Removal of the heparin-binding domain affects both the affinity of EC-SOD for and its distribution to the extracellular matrix, in which it is secreted. During the purification of human EC-SOD, the intact/cleaved ratio remains constant, suggesting that proteolytic removal of the heparin-binding domain does not occur during purification (Oury, T. D., Crapo, J. D., Valnickova, Z., and Enghild, J. J. (1996) Biochem. J. 317, 51–57). This was supported by the finding that fresh mouse tissue contains both intact and cleaved EC-SOD. To study other possible mechanisms leading to the formation of cleaved EC-SOD, we examined biosynthesis in cultured rat L2 epithelial-like cells using a pulse-chase protocol. The results of these studies suggest that the heparin-binding domain is removed intracellularly just prior to secretion. In addition, the intact/cleaved EC-SOD ratio appears to be tissue-dependent, implying that the intracellular processing event is regulated in a tissue-specific manner. The existence of this intracellular processing pathway may thus represent a novel regulatory pathway for affecting the distribution and effect of EC-SOD.

Human Procarboxypeptidase U, or Thrombin-activable Fibrinolysis Inhibitor, Is a Substrate for Transglutaminases EVIDENCE FOR TRANSGLUTAMINASE-CATALYZED CROSS-LINKING TO FIBRIN

Procarboxypeptidase U (EC 3.4.17.20) (pro-CpU), also known as plasma procarboxypeptidase B and thrombin-activable fibrinolysis inhibitor, is a human plasma protein that has been implicated in the regulation of fibrinolysis. In this study, we show that pro-CpU serves as a substrate for transglutaminases. Both factor XIIIa and tissue transglutaminase catalyzed the polymerization of pro-CpU and the cross-linking to fibrin as well as the incorporation of 5-dimethylaminonaphthalene-1-sulfonyl cadaverine (dansylcadaverine), [14C]putrescine, and dansyl-PGGQQIV. These findings show that pro-CpU contains both amine acceptor (Gln) and amine donor (Lys) residues. The amine acceptor residues were identified as Gln2, Gln5, and Gln292, suggesting that both the activation peptide and the mature enzyme participate in the cross-linking reaction. These observations imply that transglutaminases may mediate covalent binding of pro-CpU to other proteins and cell surfaces in vivo. In particular, factor XIIIa may cross-link pro-CpU to fibrin during the latter part of the coagulation cascade, thereby helping protect the newly formed fibrin clot from premature plasmin degradation. Moreover, the cross-linking may facilitate the activation of pro-CpU, stabilize the enzymatic activity, and protect the active enzyme from further degradation.

The dual nature of human extracellular superoxide dismutase: One sequence and two structures

Human extracellular superoxide dismutase (EC-SOD; EC 1.15.1.1) is a scavenger of superoxide anions in the extracellular space. The amino acid sequence is homologous to the intracellular counterpart, Cu/Zn superoxide dismutase (Cu/Zn-SOD), apart from N- and C-terminal extensions. Cu/Zn-SOD is a homodimer containing four cysteine residues within each subunit, and EC-SOD is a tetramer composed of two disulfide-bonded dimers in which each subunit contains six cysteines. The amino acid sequences of all EC-SOD subunits are identical. It is known that Cys-219 is involved in an interchain disulfide. To account for the remaining five cysteine residues we purified human EC-SOD and determined the disulfide bridge pattern. The results show that human EC-SOD exists in two forms, each with a unique disulfide bridge pattern. One form (active EC-SOD) is enzymatically active and contains a disulfide bridge pattern similar to Cu/Zn-SOD. The other form (inactive EC-SOD) has a different disulfide bridge pattern and is enzymatically inactive. The EC-SOD polypeptide chain apparently folds in two different ways, most likely resulting in different three-dimensional structures. Our study shows that one gene may produce proteins with different disulfide bridge arrangements and, thus, by definition, different primary structures. This observation adds another dimension to the functional annotation of the proteome.

Furin Proteolytically Processes the Heparin-binding Region of Extracellular Superoxide Dismutase

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SODs tissue half-life and affinity for heparin. There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D. (1999) J. Biol. Chem. 274, 14818–14822). By using mammalian cell lines, we have now determined that removal of the heparin-binding region occurs after passage through the Golgi network but before being secreted into the extracellular space. Specific protease inhibitors and overexpression of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines. A mutation in Arg213renders EC-SOD resistant to furin processing. These results indicate that furin-dependent processing of EC-SOD is important for determining the tissue distribution and half-life of EC-SOD.

The Intracellular Proteolytic Processing of Extracellular Superoxide Dismutase (EC-SOD) is a Two-step Event

Extracellular superoxide dismutase (EC-SOD) is a tetramer composed of either intact (Trp1–Ala222) or proteolytically cleaved (Trp1–Glu209) subunits. The latter form is processed intracellularly before secretion and lacks the C-terminal extracellular matrix (ECM)-binding region (210RKKRRRESECKAA222–COOH). We have previously suggested that the C-terminal processing of EC-SOD is either a one-step mechanism accomplished by a single intracellular endoproteolytic event cleaving the Glu209–Arg210 peptide bond or a two-step mechanism involving two proteinases (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D. (1999) J. Biol. Chem. 274, 14818–14822). In the latter case, an initial endoproteinase cleavage occurs somewhere in the region between Glu209 and Glu216. A carboxypeptidase specific for basic amino acid residues subsequently trims the remaining basic amino acid residues to Glu209. A naturally occurring mutation of EC-SOD substituting Arg213 for Gly enabled us to test these hypotheses. The mutation does not prevent proteolysis of the ECM-binding region but prevents a carboxypeptidase B-like enzyme from trimming residues beyond Gly213. The R213G mutation is located in the ECM-binding region, and individuals carrying this mutation have an increased concentration of EC-SOD in the circulatory system. In this study, we purified the R213G EC-SOD variant from heterozygous or homozygous individuals and determined the C-terminal residue of the processed subunit to be Gly213. This finding supports the two-step processing mechanism and indicates that the R213G mutation does not disturb the initial endoproteinase cleavage event but perturbs the subsequent trimming of the C terminus.

Human Phenotypically Distinct TGFBI Corneal Dystrophies Are Linked to the Stability of the Fourth FAS1 Domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

The high concentration of Arg213→Gly extracellular superoxide dismutase (EC-SOD) in plasma is caused by a reduction of both heparin and collagen affinities

The C-terminal region of EC-SOD (extracellular superoxide dismutase) mediates the binding to both heparin/heparan sulphate and type I collagen. A mutation (Arg213-->Gly; R213G) within this extracellular matrix-binding region has recently been implicated in the development of heart disease. This relatively common mutation affects the heparin affinity, and the concentration of EC-SOD in the plasma of R213G homozygous individuals is increased 10- to 30-fold. In the present study we confirm, using R213G EC-SOD purified from a homozygous individual, that the heparin affinity is reduced. Significantly, the collagen affinity of the R213G EC-SOD variant was similarly affected and both the heparin and collagen affinities were reduced by 12-fold. Structural analysis of synthetic extracellular matrix-binding regions suggests that the mutation alters the secondary structure. We conclude that the increased concentration of EC-SOD in the plasma of R213G carriers is caused by a reduction in both heparin and collagen affinities.

The Crystal Structure of Thrombin-activable Fibrinolysis Inhibitor (TAFI) Provides the Structural Basis for Its Intrinsic Activity and the Short Half-life of TAFIa

Mature thrombin-activable fibrinolysis inhibitor (TAFIa) is a highly unstable metallocarboxypeptidase that stabilizes blood clots by clipping C-terminal lysine residues from partially degraded fibrin. In accordance with its in vitro antifibrinolytic activity, animal studies have reported that inhibition of mature TAFI aids in the prevention of thrombosis. The level of TAFI activity is stringently regulated through (i) controlled proteolytic truncation of the zymogen (TAFI), generating the mature enzyme, TAFIa, and (ii) the short half-life of TAFIa. TAFI itself exhibits an intrinsic enzymatic activity, which is likely required to provide a baseline level of antifibrinolytic activity. The novel crystal structure presented here reveals that the active site of TAFI is accessible, providing the structural explanation for the its intrinsic activity. It also supports the notion that an “instability region” exists, in agreement with site-directed mutagenesis studies. Sulfate ions, bound to this region, point toward a potential heparin-binding site and could explain how heparin stabilizes TAFIa.