Covalent Cross-linking Of Proteins Research Articles

A Vascular Bone Collector

See related article, pages 529–537 Calcification of the arterial wall occurs in, among others, atherosclerosis and aging. The role of calcification in plaque stability is subject of ongoing discussion; media calcification in aging and diabetes increases stiffness and pulse pressure.1 It is now clear that calcification is not simply a passive process but, rather, is tightly regulated, involving induction of an osteochondrogenic phenotype in the vascular smooth muscle cells (VSMCs) and control of expression and activity of factors promoting and inhibiting calcium deposition. In this issue of Circulation Research , Johnson et al2 provide evidence that transglutaminases (Tgases), cross-linking enzymes with recently discovered vascular functions, are required for inducing the vascular calcification process. Tgases are multifunctional enzymes acting in the cell, at the cell surface, or as released enzymes. Their most prominent feature is the covalent cross-linking of proteins.3 The glutamine–lysine cross-links, either within or between proteins, result in the stabilization of extracellular matrices and the binding of signaling molecules, such as osteopontin, to the matrix but also the prolonged activation of angiotensin II receptors through dimerization.4 Cell surface Tgases are involved in cell adhesion through integrin clustering and function as coreceptors in the binding to fibronectin.5 The Tgase family consists of 9 members, of which TG1, TG2, and the coagulation factor XIIIA are expressed in the arterial wall.6 The tissue-type Tgase studied here (TG2) fulfills both cross-linking activities and G protein …

A common mechanism of PLP/DM20 misfolding causes cysteine-mediated endoplasmic reticulum retention in oligodendrocytes and Pelizaeus–Merzbacher disease

A large number of mutations in the human PLP1 gene lead to abnormal myelination and oligodendrocyte death in Pelizaeus-Merzbacher disease (PMD). Here we show that a major subgroup of PMD mutations that map into the extracellular loop region of PLP/DM20 leads to the failure of oligodendrocytes to form the correct intramolecular disulfide bridges. This leads to abnormal protein cross-links and endoplasmic reticulum retention and activates the unfolded protein response. Importantly, surface expression of mutant PLP/DM20 can be restored and the unfolded protein response can be reverted by the removal of two cysteines. Thus, covalent protein cross-links emerge as a cause, rather than as a consequence, of endoplasmic reticulum retention.

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.

Application of zero-length cross-linking to form lysozyme, horseradish peroxidase and lysozyme–peroxidase dimers: Activity and stability

A facile method for the formation of covalent bonds between protein molecules is zero-length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, the cross-linking is performed for lysozyme, peroxidase (a glycoprotein) and between lysozyme–peroxidase by the method of Simons et al. [B.L. Simons, M.C. King, T. Cyr, M.A. Hefford, H. Kaplan, Covalent cross-linking of protein without chemical reagents, Protein Sci. 2002, 11, 1558–1564]. Approximately one-third of the total lysozyme becomes cross-linked and the dimer form was the major product for both enzymes. This modification induced some changes in the kinetic properties of the dimer peroxidase, as evident by two-fold increasing of V max compared to the monomer but the enzymatic activity of cross-linked lysozyme dimer was the same as monomer. The activity of lysozyme dimer remained constant up to 10 min at 80 °C, while peroxidase activity of both monomer and dimer began to decrease after heating. The structural changes of the enzymes were investigated by circular dichroism and intrinsic fluorescence techniques. Near UV result showed lysozyme possess a compact structure in the dimer form but disruption of tertiary structure of peroxidase dimer was observed. Also conformational changes were detected and discussed by intrinsic fluorescence experiments. Effect of several metals in the formation of lysozyme dimer showed that Co 2+ is the most effective one but its effect was marginal. At the end formation of heterogeneous dimer, peroxidase–lysozyme, was achieved using this method.

Novel cross-linked enzyme–antibody conjugates for Western blot and ELISA

Covalent cross-linking of enzymes to antibodies to produce immunoconjugates for Western blot analysis and ELISAs was achieved using in vacuo cross-linking methodology [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Covalent cross-linking of proteins without chemical reagents. Protein Sci. 11, 1558.]. The advantageous feature of this methodology for producing enzyme–antibody conjugates is that the cross-linking is accomplished without the use of chemical modifying or activating reagents. This reduces the potential activity loss due to chemical modification and allows easy recovery of any free antibody or native enzyme. In vacuo cross-linking of horseradish peroxidase (HRP) to anti-rabbit immunoglobulin G (IgG) produced an enzyme-linked antibody with an improved sensitivity for antigen detection compared to a commercial conjugate prepared by conventional chemical cross-linking methods. A soluble multi-enzyme-based immunoconjugate was prepared by the in vacuo cross-linking of HRP to a high molecular weight polyglutamic acid polymer followed by the in vacuo cross-linking of a limiting amount of antibody to yield an antibody–(HRP) n –polyglutamate complex. This complex had a detection signal 100-fold greater than that of the 1:1 enzyme–antibody conjugates prepared by chemical cross-linking.

Distinct β-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

Tissue transglutaminase-induced alterations in extracellular matrix inhibit tumor invasion.

BackgroundAlterations in the extracellular matrix (ECM) can affect host-tumor interactions and tumor growth and metastasis. Tissue transglutaminase (TG2, EC 2.3.2.13), a calcium-dependent enzyme that catalyzes covalent cross-linking of proteins, can render the ECM highly stable and resistant to proteolytic degradation. So we determined whether TG2 expression in a tumor or nontumor (stroma) environment could affect the process of metastasis. Two hundred archived samples from patients with breast cancer were studied for the TG2 expression. Also, in an in vitro model the invasive behavior of MDA-MB-231 cells in the presence or absence of exogenous TG2 was determined.ResultsTumors associated with negative nodes showed significantly higher expression of TG2 in the stroma (P < 0.001). TG2 in the stroma was catalytically active, as revealed by the presence of isopeptide cross-links. Pretreatment of Matrigel with catalytically active TG2 resulted in strong inhibition of invasion of MDA-MB-231 cells through the Matrigel Transwell filters.ConclusionTG2-induced alterations in the ECM could effectively inhibit the process of metastasis. Therefore, selective induction of catalytically active TG2 at the site of tumor may offer promising approach for limiting the metastasis.

A method for the isolation of covalent DNA–protein crosslinks suitable for proteomics analysis

The covalent crosslinking of protein to DNA is a form of DNA damage induced by a number of commonly encountered agents, including metals, aldehydes, and radiation as well as chemotherapeutic drugs. DNA–protein crosslinks (DPCs) are potentially bulky and helix distorting and have the potential to block the progression of translocating protein complexes. To fully understand the induction and repair of these lesions, it will be important to identify the crosslinked proteins involved. To take advantage of dramatic improvements in instrument sensitivity that have facilitated the identification of proteins by proteomic approaches, improved methods are required for isolation of DPCs. This article describes a novel method for the isolation of DPCs from mammalian cells that uses chaotropic agents to isolate genomic DNA and stringently remove noncrosslinked proteins followed by DNase I digestion to release covalently crosslinked proteins. This method generates high-quality protein samples in sufficient quantities for analysis by mass spectrometry. In addition, the article presents a modified form of this method that also makes use of chaotropic agents for promoting the adsorption of DNA (with crosslinked proteins) to silica fines, markedly reducing the DPC isolation time and cost. These approaches were applied to radiation- and camptothecin-induced DPCs.

DNA?protein crosslinks: their induction, repair, and biological consequences

The covalent crosslinking of proteins to DNA presents a major physical challenge to the DNA metabolic machinery. DNA-protein crosslinks (DPCs) are induced by a variety of endogenous and exogenous agents (including, paradoxically, agents that are known to cause cancer as well as agents that are used to treat cancer), and yet they have not received as much attention as other types of DNA damage. This review summarizes the current state of knowledge of DPCs in terms of their induction, structures, biological consequences and possible mechanisms of repair. DPCs can be formed through several different chemistries, which is likely to affect the stability and repair of these lesions, as well as their biological consequences. The considerable discrepancy in the DPC literature reflects both the varying chemistries of this heterogeneous group of lesions and the fact that a number of different methods have been used for their analysis. In particular, research in this area has long been hampered by the inability to chemically define these lesions in intact cells and tissues. However, the emergence of proteomics as a tool for identifying specific proteins that become crosslinked to DNA has heralded a new era in our ability to study these lesions. Although there are still many unanswered questions, the identification of specific proteins crosslinked to DNA should facilitate our understanding of the down-stream effects of these lesions.

Activation of transglutaminase in μ-calpain null erythrocytes

Intracellular transglutaminases (protein–glutamine: amine γ-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, μ-calpain. Using μ-calpain null ( Capn1 −/−) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of μ-calpain. Also, the absence of μ-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of μ-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.

Covalent cross-linking of proteins without chemical reagents.

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.

Histone cross-linking by transglutaminase

Transglutaminases irreversibly catalyze covalent cross-linking of proteins by forming isopeptide bonds between peptide-bound glutamine and lysine residues. Among several transglutaminases, tissue-type transglutaminase (tTGase) is most ubiquitously found in every type of cells and tissues in animals, but its natural substrate has yet to be identified. In an attempt to identify the natural substrate for tTGase, we examined in vitro if core histones were subject to cross-linking by tTGase. We found core histone subunits, H2A and H2B, were specifically cross-linked by tTGase. The cross-linking was between either one or both glutamines at C-terminal end of H2A (-VTIA Q 104 GGVLPNT Q 112 SVLLPKKTESSKSK—C ′ end) and the first and/or third lysine from C-terminal end of H2B (-AVESEG K 116 AVTKYTSS K 125 —C ′ end). The cross-linking occurred only when these subunits were released from nucleosome but not when these were organized in nucleosome. Most interestingly, in chicken erythrocyte the cross-linked H2A–H2B was present in a significant amount. From these results, it can be proposed that tTGase-mediated cross-linking is an another form of core histone modification and it may play a role of chromatin condensation during erythrocyte differentiation.

Pressure Inactivation Kinetics of Microbial Transglutaminase from Streptoverticillium mobaraense

ABSTRACT:Microbial transglutaminase (MTGase) forms nondisulfide covalent crosslinks in proteins. Changes in activity of MTGase from Streptoverticillium mobaraense after exposure to pressure (100 to 600 MPa for 10 to 60 min, respectively) were analyzed. MTGase activity increased linearly with enzyme concentration, regardless of pressures in the range examined. The Vmax value was changed by pressure while the Km value was independent of pressure. Sodium chloride (0 to 0.86 N) apparently caused MTGase destabilization under pressure. Oscillatory pressurization (400 to 600 MPa, 25°C, 10 min/cycle, 0 to 5 cycles) resulted in a higher degree of inactivation when pressures were greater than 500 MPa. MTGase maintained 60% of its initial activity even after pressurization at 600 MPa for 60 min, indicating that MTGase was pressure‐resistant as compared to other enzymes.

A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate.

Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His(6)-Xpress). His(6)-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247-258, 2001)

Effects of sugars on whey protein isolate gelation.

Whey protein isolate (WPI) gels were prepared from solutions containing ribose or lactose at pH values ranging from 6 to 9. The gels with added lactose had no color development, whereas the gels with added ribose were orange/brown. Lactose stabilized the WPI to denaturation, which increased the time and temperature required for gelation, thus decreasing the fracture modulus of the gel compared to the gels with added ribose and the gels with no sugar added. Ribose, however, favored the Maillard reaction and covalent cross-linking of proteins, which increased gel fracture modulus. The decreased pH caused by the Maillard reaction in the gels containing ribose occurred after protein denaturation and gelation, thus having little if any effect on the gelation process.

Pastry Lift and Croissant Volume as Affected by Microbial Transglutaminase

ABSTRACT:Microbial transglutaminase forms nondisulfide covalent crosslinks in proteins and is increasingly being used in foods. We have previously demonstrated beneficial effects of microbial transglutaminase during breadmaking, which are comparable to traditional oxidizing improvers, hypothesized to act via formation of disulfide crosslinks. Transglutaminase substantially improved the lift of puff pastry. It also had a dramatic effect on the volume of yeasted croissants made with both white flour and a blend of wholemeal and white flour. Furthermore, these effects were preserved after the pastry and croissant doughs had undergone frozen storage for periods of up to 90 d. Transglutaminase, therefore, offers a potential solution to the problem of pastry and croissant dough deterioration on frozen storage.

A Urokinase Receptor-associated Protein with Specific Collagen Binding Properties

The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high molecular weight urokinase receptor-associated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices.

Transglutaminase Type 1 and Its Cross-linking Activity Are Concentrated at Adherens Junctions in Simple Epithelial Cells

Transglutaminase type 1 was identified as a tyrosine-phosphorylated protein from the isolated junctional fraction of the mouse liver. This enzyme was reported to be involved in the covalent cross-linking of proteins in keratinocytes, but its expression and activity in other cell types have not been examined. Northern blotting revealed that transglutaminase type 1 was expressed in large amounts in epithelial tissues (lung, liver, and kidney), which was also confirmed by immunoblotting with antibodies raised against mouse recombinant protein. Immunoblotting of the isolated junctional fraction revealed that transglutaminase type 1 was concentrated in the fraction not only as a 97-kDa form but also as forms of various molecular masses cross-linked to other proteins. In agreement with this finding, endogenous transglutaminase type 1 was immunofluorescently colocalized with E-cadherin in cultured simple epithelial cells. In the liver and kidney, immunoelectron microscopy revealed that transglutaminase type 1 was concentrated, albeit not exclusively, at cadherin-based adherens junctions. Furthermore, by in vitro and in vivo labeling, transglutaminase cross-linking activity was also shown to be concentrated at intercellular junctions of simple epithelial cells. These findings suggested that the formation of covalently cross-linked multimolecular complexes by transglutaminase type 1 is an important mechanism for maintenance of the structural integrity of simple epithelial cells, especially at cadherin-based adherens junctions.

Dough Properties and Crumb Strength of White Pan Bread as Affected by Microbial Transglutaminase

ABSTRACTMicrobial transglutaminase forms nondisulfide covalent crosslinks in proteins and is being used in foods. This enzyme may produce beneficial effects during breadmaking that are comparable to traditional oxidizing improvers, hypothesized to act via formation of disulfide crosslinks. Transglutaminase greatly improved the crumb strength of baked loaves and provides a potential solution to a common consumer complaint. Transglutaminase also reduced the required work input and substantially improved the water absorption of the dough. Each of these effects would lower processing costs for commercial baking

Tissue transglutaminase is not increased during apoptosis of HT-1080 human fibrosarcoma cells

Tissue transglutaminase (tTGase), a cytosolic enzyme which catalyzes the covalent cross-linking of proteins, is thought to be involved in the apoptosis. Here, we tested whether tTGase is involved during HT-1080 fibrosarcoma cell apoptosis induced by the YIGSR (Tyr-Ile-Gly-Ser-Arg) peptide. This sequence is derived from the laminin alpha1 chain, and its potency is increased by the formation of a 16mer polymerization using a lysine tree structure. Cells were treated with several different concentrations of Ac-Y 16 for 16 hours, and apoptosis was increased in dose-dependent manner. When assayed by incorporation of [14C] putrescine into succinylated casein, total transglutaminase activity was decreased in parallel with the change in the number of attached cells. Western blot analysis using polyclonal antibody against tTGase showed that the tTGase protein level had not been significantly changed when equal amounts of the protein were applied. To confirm this result, we induced apoptosis of these cells by coating the tissue culture plates with non-adhesive poly-hydroxyethyl methacrylate (HEMA). Western blot analysis showed that the tTGase protein level did not change during this process of apoptosis. Although it has been suggested that tTGase is involved in the process of apoptosis of various cells in vitro and in vivo, our data demonstrate that tTGase is not involved in the process of apoptosis of HT-1080 human fibrosarcoma cell induced by either Ac-Y 16 or a non-adhesive culture surface.