Lysyl Residues Research Articles

Crystal Structure of the 2-Oxoglutarate- and Fe(II)-Dependent Lysyl Hydroxylase JMJD6

Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine–serine-rich domains of RNA-splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than Nɛ-demethylation, as for analogous enzymes.

Synthesis and Characterization of Biosuperabsorbent Based on Ovalbumin Protein

A biosuperabsorbent (Bio-SAP) hydrogel from ovalbumin (egg protein) was synthesized via modification with an acylating reagent and a bifunctional crosslinker, and its swelling behavior was investigated. The protein was acylated using ethylenediaminetetraacetic dianhydride (EDTAD), and then crosslinked by glutaraldehyde and dried. Bio-SAP provided through this method includes modification of lysyl residues in the unfolded protein by adding one or more hydrophilic carboxyl groups to increase the hydrophilicity of protein. The water binding capacity was measured in deionized water, 0.9% NaCl solution and synthetic urine, which under the best conditions were 296, 64 and 56 g/g after 24 h, respectively. In addition, the effects of EDTAD/protein ratio on the chemical modification of the protein, the various chemical neutralization agents, pH sensitivity and ionic strength, as well as temperature and particle size on the water absorption capacity with and without load and its kinetic were also investigated.

Gas-Phase Fragmentation Characteristics of Benzyl-Aminated Lysyl-Containing Tryptic Peptides

The fragmentation characteristics of peptides derivatized at the side-chain epsilon-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C(zeta)-N(epsilon) bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n - 1)(+)-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzyl-modified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary epsilon-amino group of the benzyl-derivatized lysyl side chain. If the secondary epsilon-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary epsilon-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.

Model reactions for insect cuticle sclerotization: Participation of amino groups in the cross-linking of Manduca sexta cuticle protein MsCP36

Current theories of sclerotization center on protein cross-linking and dehydration as major factors in the hardening and stability of the insect cuticle. Several studies have reported the identification of catechol-amino acid adducts from sclerotizing cuticle involving histidine, lysine, and tyrosine, though there have been no reports of a catechol linked between two amino acid residues. Previously, we reported an in vitro model system for sclerotization and observed that stable protein oligomers were formed, presumably through cross-links with oxidized catecholamines [Insect Biochem. Mol. Biol. (2006) 36, 353–365]. Using site-directed mutagenesis we created a mutant lacking histidine, rMsCP36(H65A/H178A), to investigate the possible involvement of the two histidine residues of MsCP36 in cross-linking. Surprisingly, this alteration had little or no effect on the formation of protein oligomers as determined by SDS-PAGE analysis. Blocking of the free amino groups in lysyl side chains and the amino-terminus by succinylation diminished, but did not eliminate, cross-linking of either rMsCP36 or rMsCP36(H65A/H178A). We also examined the possibility that cross-linking was due to intermolecular dityrosine linkages. Immunoblot analysis utilizing a monoclonal antibody known to recognize peptidyl dityrosine indicated that dityrosyl cross-links were present. Taken together, these results indicate that lysyl residues are important for the cross-linking of the cuticle protein rMsCP36, but that additional residues other than histidine can also contribute.

Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity

The human immunodeficiency virus type 1 aspartyl protease (HIV-1 PR) is a homodimeric aspartyl endopeptidase that is required for virus replication. HIV-1 PR was shown to act in vitro as acyl-donor and -acceptor for both guinea pig liver transglutaminase (TG, EC 2.3.2.13) and human Factor XIIIa. These preliminary evidences suggested that the HIV-1 PR contains at least three TG-reactive glutaminyl and one lysyl residues. We report here that the incubation of HIV-1 PR with TG increases its catalytic activity. This increase is dependent upon the time of incubation, the concentration of TG and the presence of Ca 2+. Identification of ε-(γ-glutamyl)lysine in the proteolytic digest of the TG-modified HIV-1 PR suggested intramolecular covalent cross-linking of this protease which may promote a non-covalent dimerization and subsequent activation of this enzyme via a conformational change. This hypothesis is supported by the observation that the TG-catalyzed activation of HIV-1 PR was completely abolished by spermidine (SPD) which acts as a competitive inhibitor of ε-(γ-glutamyl)lysine formation. Indeed, in the presence of 1 mM SPD the formation of the isopeptide was decreased of about 80%. The main products of the TG-catalyzed modification of HIV-1 PR in the presence of SPD were N 1- mono(γ-glutamyl)SPD and N 8- mono(γ-glutamyl)SPD. Negligible amount of N 1,N 8- bis(γ-glutamyl)SPD were found. The significance of these results is discussed with respect to the activation of the protease by post-translational modification and design of potential inhibitors.

PH-Stability and Thermal Properties of Microbial Transglutaminase-Treated Whey Protein Isolate

Whey protein isolate (WPI) was treated to various extents using microbial transglutaminase (MTGase) and changes in pH-stability and thermal stability of its protein components were investigated. The MTGase treatment significantly increased the denaturation temperature (T(d)) of beta-lactoglobulin in WPI, from 71.84 degrees C in the untreated sample to 78.50 degrees C after 30 h of incubation with MTGase. The enthalpy change of denaturation of WPI did not change upon cross-linking, indicating that the increase in T(d) was primarily due to covalent cross-linking and not due to an increase in nonpolar interactions within the protein. The surface hydrophobicity (S(o)) of the protein decreased upon cross-linking; however, this decrease was not due to burial of the surface hydrophobic cavities in the protein interior, but due to occlusion of the hydrophobic cavities to the fluorescent probes. Fluorescence emission and circular dichroism spectroscopic analyses revealed no major changes in the secondary and tertiary conformations as a result of cross-linking. However, unlike native WPI, the cross-linked protein exhibited a U-shaped pH-stability profile with maximum turbidity at pH 4.0-4.5. The study revealed that even though enzymatic cross-linking significantly improved the T(d) of beta-lactoglobulin in WPI without causing major structural changes, a reduction in the hydrophilic-hydrophobic balance of the protein surface as a result of elimination of the positive charge on lysyl residues caused precipitation at pH 4.0-4.5.

Interaction of l-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions

The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO/VAP-1. The present work reports the kinetics of the interaction of l-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since d-lysine, l-lysine ethyl ester and ε-acetyl- l-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H 2O 2, formed during the oxidation of a physiological SSAO substrate, yet to be identified.

Physio-pathological roles of transglutaminase-catalyzed reactions

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze post-translational modifications of proteins. The main activity of these enzymes is the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted /crosslinked adducts) or -OH groups (to form ester linkages). In the absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. The TG enzymes are also capable of catalyzing other reactions important for cell viability. The distribution and the physiological roles of TG enzymes have been widely studied in numerous cell types and tissues and their roles in several diseases have begun to be identified. "Tissue" TG (TG2), a member of the TG family of enzymes, has definitely been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology: i.e. celiac disease (CD). TG activity has also been hypothesized to be directly involved in the pathogenetic mechanisms responsible for several other human diseases, including neurodegenerative diseases, which are often associated with CD. Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, supranuclear palsy, Huntington's disease and other recently identified polyglutamine diseases, are characterized, in part, by aberrant cerebral TG activity and by increased cross-linked proteins in affected brains. In this review, we discuss the physio-pathological role of TG-catalyzed reactions, with particular interest in the molecular mechanisms that could involve these enzymes in the physio-pathological processes responsible for human neurodegenerative diseases.

Role of the Transglutaminase Enzymes in the Nervous System and their Possible Involvement in Neurodegenerative Diseases

Transglutaminases are a large family of related and ubiquitous enzymes which catalyze the cross linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted /crosslinked adducts) or -OH groups (to form ester linkages). In absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. These enzymes are also capable of catalyzing other reactions important for cell viability. The distribution and the physiological roles of human transglutaminases have been widely studied in numerous cell types and tissues and their roles in several diseases have begun to be identified. Recently, "tissue" transglutaminase (TG2) has been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology, celiac disease (CD). Transglutaminase activity has also been hypothesized to be directly involved in the pathogenetic mechanisms responsible for several human neurodegenerative diseases, which are characterized in part by aberrant cerebral transglutaminase activity and by increased cross-linked proteins in affected brains, such as Alzheimer's disease (AD), Parkinson's disease (PD), supranuclear palsy, Huntington's disease (HD) and the other recently identified polyglutamine diseases, and others. In this review we discuss the biological role of the transglutaminases in the nervous system, with particular interest in the molecular mechanisms, which could involve these enzymes in the pathophysiological processes responsible for human neurodegenerative diseases.

CHARACTERIZATION AND COMPARISON OF COLLAGENS EXTRACTED FROM THE DIGESTIVE TRACT AND SKIN OF A JAPANESE AMBERJACK SERIOLA QUINQUERADIATA

ABSTRACT Collagen was extracted from the digestive tract and skin of a Japanese amberjack (Seriola quinqueradiata) by acid extraction and limited pepsin digestion. The amounts of collagen solubilized from the digestive tract were smaller than those from the skin. Based on the solubility in NaCl solution, electrophoretic and peptide map patterns, and amino acid composition, the main digestive tract collagen was identified as type I, having characteristics different from those of the body wall collagen in cyclostome intestine. Further, the degree of hydroxylation of prolyl and lysyl residues in the type I collagen of the digestive tract is significantly higher than that of the skin. Collagen preparations from the digestive tract have a higher ratio of type V collagen than those from the skin. Hence, the digestive tract collagen differs from that in the skin in the degree or property of intermolecular crosslinking, posttranslational modification, and molecular species composition. PRACTICAL APPLICATIONS Partial hydrolyzate of gelatin, in other word collagen peptide, has gained popularity as a food ingredient, as it has been suggested to have health benefits, such as improvement of skin and joint conditions. Recently, attention toward collagen derived from marine origin such as fish skin increased because of the outbreak of bovine spongiform encephalopathy. Large amounts of the digestive tract, stomach, intestine and adhesion tissues are generated by fishery industries and most of them are by-products of low value. Although these organs are also rich in collagen, the collagen in fish digestive tract has not been characterized. The present study demonstrates that the collagen in digestive tract differs from the skin collagen in the solubility, posttranslational modification and molecular species composition. These facts suggest that modified collagen peptides might be obtained from the digestive tract.

Synthesis and structural characterization of carboxyethylpyrrole-modified proteins: mediators of age-related macular degeneration

Protein modifications in which the ε-amino group of lysyl residues is incorporated into a 2-(ω-carboxyethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angiogenesis into the retina (‘wet AMD’) and geographic retinal atrophy (‘dry AMD’). Blood levels of CEPs are biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting AMD, for example, through the activation of B- and T-cells, interaction with receptors, or binding with complement proteins, we developed an efficient synthesis of CEP derivatives, that is especially effective for proteins. The structures of tryptic peptides derived from CEP-modified proteins were also determined. A key finding is that 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester reacts with primary amines to provide 9-fluorenylmethyl esters of CEP-modified proteins that can be deprotected in situ with 1,8-diazabicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-modifications with a wide variety of CEP:protein ratios is readily achieved using this strategy.

Investigation on the Scavenging Mechanism of 1,4‐Dicarbonyls by Pyridoxamine: A Density Functional Theory Study

AbstractThe dicarbonyl compound 4‐oxopentanal (OPA) is one of the reactive carbonyl species formed in a variety of metabolic reactions in vivo, which could react with lysyl residues to form pyrrolic compounds and exhibit severe toxicity. Pyridoxamine (PM) could effectively scavenge such reactive carbonyl compounds. The reaction mechanism of PM with OPA was investigated by density functional theory (DFT) B3LYP at 6‐31G∗︁ basis level together with MP2/6‐31+G∗︁∗︁ single point calculation on the stationary points. The results indicated that this reaction could be achieved through two continuous steps. Firstly, PM and OPA would generate a tetrahydropyrrole compound via two possible reaction pathways. Subsequently, two molecules of water would be removed to achieve the final pyrrole‐type compound through two parallel paths. In addition, it is possible to form an imine intermediate in this reaction, which seems more like a by‐product from our calculation. In the end, the steric hindrance of the acetyl group was discussed. All the results agreed with experiment very well.

Use of pH and Kinetic Isotope Effects To Establish Chemistry as Rate-Limiting in Oxidation of a Peptide Substrate by LSD1

The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the 21 N-terminal residues of histone H3, with a dimethylated lysyl residue at position 4. At pH 7.5, the rate constant for flavin reduction, k(red), equals k(cat), establishing the reductive half-reaction as rate-limiting at physiological pH. Deuteration of the lysyl methyls results in identical kinetic isotope effects of 3.1 +/- 0.2 on the k(red), k(cat), and k(cat)/K(m) values for the peptide, establishing C-H bond cleavage as rate-limiting with this substrate. No intermediates between oxidized and reduced flavin can be detected by stopped-flow spectroscopy, consistent with the expectation for a direct hydride transfer mechanism. The k(cat)/K(m) value for the peptide is bell-shaped, consistent with a requirement that the nitrogen at the site of oxidation be uncharged and that at least one of the other lysyl residues be charged for catalysis. The (D)(k(cat)/K(m)) value for the peptide is pH-independent, suggesting that the observed value is the intrinsic deuterium kinetic isotope effect for oxidation of this substrate.

Effects of Heating and Glycation of β-Lactoglobulin on Its Recognition by IgE of Sera from Cow Milk Allergy Patients

beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.

Characterization of a Naturally Occurring Mutation (Ile-12 to Thr) Close to Prosthetic Group FAD in Human Dihydrolipoamide Dehydrogenase

It catalyzes the reoxidation of the dihydrolipoyl prosthetic group attached to the lysyl residue(s) of the acyltransferase components of the three α-keto acid dehydrogenase complexes and to the hydrogen-carrier protein of the glycine cleavage system. It is a homodimeric flavoenzyme containing one FAD at each subunit.

Transglutaminases and neurodegeneration

Transglutaminases (TGs) are Ca2+-dependent enzymes that catalyze a variety of modifications of glutaminyl (Q) residues. In the brain, these modifications include the covalent attachment of a number of amine-bearing compounds, including lysyl (K) residues and polyamines, which serve to either regulate enzyme activity or attach the TG substrates to biological matrices. Aberrant TG activity is thought to contribute to Alzheimer disease, Parkinson disease, Huntington disease, and supranuclear palsy. Strategies designed to interfere with TG activity have some benefit in animal models of Huntington and Parkinson diseases. The following review summarizes the involvement of TGs in neurodegenerative diseases and discusses the possible use of selective inhibitors as therapeutic agents in these diseases.

Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry

The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNAIle, the 2',3'-dialdehyde derivative of tRNAIle was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNAIle aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNAIleox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNAIle and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence 601KMSKS605. Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [32P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.

1H NMR metabonomics can differentiate the early atherogenic effect of dairy products in hyperlipidemic hamsters

Diet is an important environmental factor modulating the onset of atherosclerosis. The aim of this study was to evaluate the effects of different dairy-based food products on early atherogenesis using both conventional and metabonomic approaches in hyperlipidemic hamsters. The hamsters received up to 200g/kg of fat as anhydrous butter or cheese made from various milk fats or canola-based oil (CV), in addition to a non-atherogenic low-fat diet. Aortic cholesteryl ester loading was considered to be an early atherogenic point, and metabolic changes linked to atherogenesis were measured using plasma 1H NMR-based metabonomics. The lowest atherogenicity was obtained with the plant-oil cheese diet, followed by the dairy fat cheese diet, while the greatest atherogenicity was observed with the butter diet (P<0.05). Disease outcome was correlated with conventional plasma biomarkers (total cholesterol, triglycerides, LDL cholesterol, R2=0.42–0.60). NMR plasma metabonomics selectively captured part of the diet-induced metabotypes correlated with aortic cholesteryl esters (R2=0.63). In these metabotypes, VLDL lipids, cholesterol, and N-acetylglycoproteins (R2 range: 0.45–0.51) were the most positively correlated metabolites, whereas a multimetabolite response at 3.75ppm, albumin lysyl residues, and trimethylamine-N-oxide were the most negatively correlated metabolites (R2 range: 0.43–0.63) of the aortic cholesteryl esters. Collectively, these metabolites predicted 89% of atherogenic variability compared to the 60% predicted by total plasma cholesterol alone. In conclusion, we show that the food environment can modulate the atherogenic effect of dairy fat. This proof-of-principle study demonstrates the first use of plasma metabonomics for improving the prognosis of diet-induced atherogenesis, revealing novel potential disease biomarkers.

Ion‐specific modulation of protein interactions: Anion‐induced, reversible oligomerization of a fusion protein

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s(w)) and hydrodynamic diameter (D(H)) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s(w) and D(H), reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D(H) and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M(z)) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I(-) > Br(-) > Cl(-) > F(-)) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the ..xxCTRWPWMC..xxxCTRWPWMCxx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.

Abstract 3623: Molecular Determinants Of Endocytosis And Tyrosine Phosphorylation Within The Cytoplasmic Tail Of Human Syndecan-1, A Receptor For Remnant Lipoproteins

HomeCirculationVol. 118, No. suppl_18Abstract 3623: Molecular Determinants Of Endocytosis And Tyrosine Phosphorylation Within The Cytoplasmic Tail Of Human Syndecan-1, A Receptor For Remnant Lipoproteins Free AccessMeeting ReportAboutSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessMeeting ReportBasic ScienceVascular Biology (Atherosclerosis/Hemostasis/Lipid Metabolism)Abstract 3623: Molecular Determinants Of Endocytosis And Tyrosine Phosphorylation Within The Cytoplasmic Tail Of Human Syndecan-1, A Receptor For Remnant Lipoproteins keyang Chen, Ming-lin Liu and Kevin J Williams keyang Chenkeyang Chen Thomas Jefferson Univ, Philadelphia, PA Search for more papers by this author , Ming-lin LiuMing-lin Liu Thomas Jefferson Univ, Philadelphia, PA Search for more papers by this author and Kevin J WilliamsKevin J Williams Thomas Jefferson Univ, Philadelphia, PA Search for more papers by this author Originally published28 Oct 2008https://doi.org/10.1161/circ.118.suppl_18.S_453Circulation. 2008;118:S_453AbstractWe previously reported that the syndecan-1 heparan sulfate proteoglycan directly mediates cellular uptake of model remnant lipoproteins through a novel endocytic pathway distinct from coated pits. Using a chimera, FcR-Synd, that consists of an IgG Fc receptor ectodomain linked to the transmembrane and cytoplasmic regions of syndecan-1, we found that efficient endocytosis is triggered by clustering of syndecan or the chimera. Syndecan clustering would be expected upon binding a multivalent ligand, such as a remnant lipoprotein. Clustering of the syndecan transmembrane and cytoplasmic domains causes their rapid movement into cholesterol-rich, detergent-insoluble, membrane rafts, and then the actual uptake into the cell requires recruitment of tyrosine kinases and the actin cytoskeleton (Biochem J 351:607, 2000Google Scholar). We now sought to identify the molecular determinants. We performed alanine scanning mutagenesis of 4 –5 consecutive residues at a time, to cover the entire syndecan cytoplasmic tail within the FcR-Synd chimera (excepting the first cytoplasmic residue, R, which was required for export to the cell surface). The parent and mutant constructs were each expressed in McArdle 7777 hepatoma cells. All alanine scanning mutants moved into rafts upon clustering. Replacement of a highly conserved, juxtamembrane motif, MKKK, with 4 alanines was the only mutation that blocked efficient endocytosis after clustering. Despite the presence of conserved lysyl residues, we did not find ubiquitinylated FcR-Synd without or with clustering. Clustering did, however, induce tyrosine phosphorylation of FcR-Synd, and this effect was absent from the mutant lacking the MKKK motif. Syndecan-mediated endocytosis was recently reported to activate extracellular signal-related kinases (ERK1/2), perhaps through the ectodomain (J Cell Biochem 99:936, 2006Google Scholar). Here, we found that engagement of the FcR-Synd chimera activated ERK, implicating the syndecan cytoplasmic tail. Furthermore, the ERK inhibitor U0126 blocked efficient endocytosis of FcR-Synd upon clustering. Overall, our results identify a novel motif, MKKK, in triggering tyrosine phosphorylation of the syndecan cytoplasmic tail and then endocytosis via rafts, in a process dependent on ERK signaling. Previous Back to top Next FiguresReferencesRelatedDetailsCited By Chen K and Williams K (2013) Molecular Mediators for Raft-dependent Endocytosis of Syndecan-1, a Highly Conserved, Multifunctional Receptor, Journal of Biological Chemistry, 10.1074/jbc.M112.444737, 288:20, (13988-13999), Online publication date: 1-May-2013. October 28, 2008Vol 118, Issue suppl_18 Advertisement Article InformationMetrics https://doi.org/10.1161/circ.118.suppl_18.S_453 Originally publishedOctober 28, 2008 Advertisement