Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5′-terminal untranslated region (UTR) of 60 bp and a 3′-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys⁵³, Cys¹²⁸, Cys¹⁴⁴, Cys¹⁵²) involved in the formation of disulfides bridges and the potential Ca²⁺/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca²⁺, and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei.     

Molecular cloning,characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper,Epinephelus coioides

C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell–cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys⁷¹, Cys¹⁵², Cys¹⁶⁷, Cys¹⁷⁵) involved in the formation of disulphide bridges and the potential Ca²⁺/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca²⁺-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca²⁺-dependent manner.     

cDNA sequence and tissues expression analysis of lipoprotein lipase from common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> Var. Jian)

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult common carp (Cyprinus carpio Var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 2,411 bp long with a 1,524 bp open reading frame (ORF) encoding 507 amino acids. This amino acid sequence contains two structural regions: N-terminus (24–354 residues) and C-terminus (355–507 residues). Before N-terminus, 1–23 residues is signal peptide, 6–23 residues is transmembrance helix. At N-terminus, some conversed functional sites were found, including two N-linked glycosylation sites Asn⁴¹ and Asn⁸⁸; one catalytic triad Ser¹⁷⁴, Asp¹⁹⁸ and His²⁸³; one conserved heparin-binding site Arg³²¹ to Arg³²⁴ (RKNR); eight cysteines residues Cys⁶⁹ and Cys⁸², Cys²⁵⁸ and Cys²⁸¹, Cys³⁰⁶ and Cys³²⁵, Cys³¹⁷ and Cys³²⁰ which are involved in four disulfide bridges; one polypeptide “lid” that participates in substrate specificity. At C-terminus, Asn⁴⁰¹ is another N-linked glycosylation site, and Trp⁴³⁴ and Trp⁴³⁵ (WW) is lipid-binding site. The amino acid sequence has a high similarity, and shows similar structural features to LPL of other species. Tissue distribution of LPL mRNA in liver, head kidney, mesenteric adipose tissue, heart and white muscle of common carp was analyzed by semi-quantitative RT-PCR method using β-actin gene as internal control. The result showed that the expressions of LPL mRNA were detected in all examined tissues of common carp. The expression levels of LPL in the mesenteric adipose tissue was highest among these tissues, following in liver and head kidney, and the lowest expression was found in heart and white muscle.     

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys⁷–Cys⁹⁴, Cys⁷²–Cys⁷⁷, Cys¹⁰¹–Cys¹²⁸ and Cys¹⁷⁹–Cys²²⁶. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.     

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

Biochemical evidence that regulation of Ero1β activity in human cells does not involve the isoform-specific cysteine 262

In the ER (endoplasmic reticulum) of human cells, disulfide bonds are predominantly generated by the two isoforms of Ero1 (ER oxidoreductin-1): Ero1α and Ero1β. The activity of Ero1α is tightly regulated through the formation of intramolecular disulfide bonds to help ensure balanced ER redox conditions. Ero1β is less tightly regulated, but the molecular details underlying control of activity are not as well characterized as for Ero1α. Ero1β contains an additional cysteine residue (Cys²⁶²), which has been suggested to engage in an isoform-specific regulatory disulfide bond with Cys¹⁰⁰. However, we show that the two regulatory disulfide bonds in Ero1α are likely conserved in Ero1β (Cys⁹⁰–Cys¹³⁰ and Cys⁹⁵–Cys¹⁰⁰). Molecular modelling of the Ero1β structure predicted that the side chain of Cys²⁶² is completely buried. Indeed, we found this cysteine to be reduced and partially protected from alkylation in the ER of living cells. Furthermore, mutation of Cys¹⁰⁰–but not of Cys²⁶²–rendered Ero1β hyperactive in cells, as did mutation of Cys¹³⁰. Ero1β hyperactivity induced the UPR (unfolded protein response) and resulted in oxidative perturbation of the ER redox state. We propose that features other than a distinct pattern of regulatory disulfide bonds determine the loose redox regulation of Ero1β relative to Ero1α.     

Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.     

Localization of disulfide bonds in α1-acid glycoprotein and their effect on the ability of the protein to interact with ethidium bromide

α1-Acid glycoprotein (orosomucoid) was purified from the human and murine blood sera using phenol deproteinization. As opposed to the murine protein, the human orosomucoid bound the fluorescent dye ethidium bromide but lost this ability after treatment with β-mercaptoethanol, which breaks disulfide bonds. Disulfide bonds between the Cys²³ and Cys¹⁶⁵ residues of the human orosomucoid and between the Cys⁹¹ and Cys¹⁸⁴ residues of the murine orosomucoid were identified.     

Sequential Comparison of Peptides Containing Half-cystine Residues from Ovalbumins of Six Avian Species

Hen ovalbumin contains one cystine disulfide (Cys⁷³-Cys¹²⁰) and four cysteine sulfhydryl groups (Cys¹¹,Cys³⁰,Cys³⁶⁷, and Cys³⁸²) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys¹¹, Cys³⁶⁷, and Cys³⁸²); and two for Pekin duck, mallard duck, and Emden goose (Cys¹¹ and Cys³³¹). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys⁷³-Cys¹²⁰). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.     

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low K_m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K_m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys¹⁶⁰ of LAT1 and Cys¹¹⁰ of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl₂ with a K_i of 0.56 ± 0.11 μM. The inhibitory effect of HgCl₂ for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl₂ for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys⁸⁸ and Cys⁴³⁹, altered amino acid transport. The V_max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys⁴³⁹ residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Formation of Two Intramolecular Disulfide Bonds Is Necessary for ApoA-I-dependent Cholesterol Efflux Mediated by ABCA1

ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formation, apoA-I binding, and HDL formation in these mutants. From the single cysteine replacements, two cysteines (Cys⁷⁵ and Cys³⁰⁹) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys¹⁴⁷⁷ was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys¹⁴⁶³ and Cys¹⁴⁶⁵, impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys⁷⁵, Cys³⁰⁹, Cys¹⁴⁶³, Cys¹⁴⁶⁵, and Cys¹⁴⁷⁷) are involved in these functions of ABCA1. Trypsin digestion experiments suggested that one disulfide bond is not sufficient and that two intramolecular disulfide bonds (between Cys⁷⁵ and Cys³⁰⁹ in ECD1 and either Cys¹⁴⁶³ or Cys¹⁴⁶⁵ and Cys¹⁴⁷⁷ in ECD2) are required for ABCA1 to be fully functional.Maintenance of cellular cholesterol homeostasis is important for normal human physiology; its disruption can lead to a variety of pathological conditions, including cardiovascular disease (1). ABCA1 (ATP-binding cassette protein A1), a key factor in cholesterol homeostasis, mediates the secretion of cellular free cholesterol and phospholipids to an extracellular acceptor, apoA-I, to form high density lipoprotein (HDL)² (2, 3). HDL formation is the only known pathway for the elimination of excess cholesterol from peripheral cells. Defects in ABCA1 cause Tangier disease (4–6), in which patients have a near absence of circulating HDL, prominent cholesterol ester accumulation in tissue macrophages, and premature atherosclerotic vascular disease (1, 7).ABCA1 is a member of the ABCA subclass of ABC transporters, which contain the basic architecture of the “full-length” ABC transporters organized into two tandemly arranged halves. Each half contains several transmembrane α-helices (TMs), which provide a translocation pathway, followed by a cytoplasmic nucleotide-binding domain, which hydrolyze ATP. In the case of “half-size” ABC transporters, such as ABCG1, ABCD1, TAP1/TAP2 (transporter associated with antigen processing), and the bacterial homolog Sav1866, they dimerize to form the full transporter. Crystallographic analysis of the bacterial homolog Sav1866 revealed that the TMs of one subunit are closely related to the TMs of the other subunit, forming two “wings” in an outward-facing conformation (8).When ABCA1 is partially digested by trypsin, ABCA1 is cleaved at site A, just C-terminal to TM6, and at site B, just N-terminal to TM7, to produce four fragments of 170 and 150 kDa and subsequently of 125 and 110 kDa (Fig. 1A) (9). When these fragments are analyzed by SDS-PAGE under nonreducing conditions, they co-migrate with undigested ABCA1. These results suggest that the N- and C-terminal halves of ABCA1 are connected by disulfide bond(s), as reported for ABCA4 (ABCR) (10). The ABCA subclass is distinguished from other ABC transporter subclasses by the presence of large extracellular domains (ECDs) (Fig. 1A) (11, 12). ECD1 and ECD2 of ABCA1 contain nine and five cysteine residues, respectively, and each connecting loop between TM5 and TM6 and between TM11 and TM12 contains a cysteine residue. These cysteine residues were assigned numbers, C1 to C16, based on their distance from the N terminus (Fig. 1A). These cysteine residues are well conserved among ABCA1, ABCA4, and ABCA7 (Fig. 1B). All of the cysteine residues in ECD1 are conserved between ABCA1 and ABCA4, and seven cysteine residues (except C4 and C5) are conserved in ABCA7. All five of the cysteine residues in ECD2 are conserved between ABCA1 and ABCA4, and three cysteine residues (except C11 and C12) are conserved in ABCA7. Because ABCA7, like ABCA1, mediates apoA-I-dependent lipid efflux (13, 14), conserved cysteine residues might be important for its function. Indeed, the Tangier disease mutation C1477R has been reported to abolish apoA-I binding and HDL formation (15–17), and several missense mutations in cysteine residues within ECD1 (C54Y, C75G) and ECD2 (C1488R, C1490Y) of ABCA4 have been linked to Stargardt disease (18–21). It remains unclear, however, whether disulfide bond formation is important for the proper folding and/or the functions of ABCA subclass proteins.Open in a separate windowFIGURE 1.Structural features of ABCA1. A, topological model for human ABCA1. ABCA1 consists of 12 transmembrane α-helices (TM1–TM12) and two large ECDs. ECD1 and ECD2 contain nine and five cysteine residues, respectively, and each connecting loop between TM5 and TM6 and between TM11 and TM12 contains a cysteine residue. These cysteine residues were assigned numbers, from C1 to C16, based on their distance from the N terminus. ABCA1 is cleaved at sites A and B by limited trypsin digestion. B, amino acid sequence alignment of ECDs of human ABCA1, ABCA4, and ABCA7. Conserved cysteine residues are indicated in black boxes.In this study, we analyzed which cysteine residues are involved in disulfide bond formation and examined whether disulfide bond formation is necessary for the functions of ABCA1. Cysteine substitution experiments suggested that two disulfide bonds are formed between C2 and C6 in ECD1 and between either C13 or C14 and C15 in ECD2 and that this two-disulfide bond formation is necessary for apoA-I-dependent cholesterol efflux by ABCA1.     

Redox Regulation of Transglutaminase 2 Activity

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys²³⁰, Cys³⁷⁰, and Cys³⁷¹ that is involved in oxidative inactivation of TG2. Within this triad, Cys³⁷⁰ was found to participate in disulfide bonds with both Cys²³⁰ and its neighbor, Cys³⁷¹. Notably, Ca²⁺ was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys²³⁰ appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys³⁷⁰–Cys³⁷¹ through formation of the Cys²³⁰–Cys³⁷⁰ disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys²³⁰ is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.     

rCNT2 extracellular cysteines,Cys615 and Cys649, are important for maturation and sorting to the plasma membrane

rCNT2 is a purine-preferring concentrative nucleoside transporter implicated in the regulation of extracellular adenosine levels and purinergic signaling. This study addressed the analysis of the CNT2 C-terminus tail as a domain likely to be implicated in transporter sorting. The topological mapping of this segment revealed that Cys⁶¹⁵ and Cys⁶⁴⁹ are important residues for the proper trafficking of CNT2 to the plasma membrane. The inhibition of protein disulfide isomerase (PDI) and ER glycosidase I and II impaired rCNT2 trafficking to the cell surface, similarly to Cys⁶¹⁵ and Cys⁶⁴⁹ mutants. The present work suggests these two cysteine residues are relevant for the proper sorting of the transporter and its functional performance.     

Disulfide Linkage and Structure of Highly Stable Yeast-derived Virus-like Particles of Murine Polyomavirus

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys¹¹⁵–Cys²⁰, Cys¹²–Cys²⁰, Cys¹⁶–Cys¹⁶, Cys¹²/ Cys¹⁶–Cys¹¹⁵, and Cys²⁷⁴–Cys²⁷⁴), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Fluorescence study of neurohypophyseal hormones and their analogues

Analogues of arginine-vasopressin (AVP) in which substitution of the proline residue in position 7 (by either sarcosine or N-methylalanine) combined with replacement of the cysteine residue in position 1 were the subject of a fluorescence and molecular mechanics study. We obtained two groups of analogues: selective antidiuretic agonists (cysteine or β-mercaptopropionic acid in position 1) and pressor and uterotonic antagonists (deaminopenicillamine or β-mercapto-β,β-cyclopentamethylene- propionic acid in position 1). Using frequency-domain measurements of fluorescence resonance energy transfer (FRET) we estimated the distance distribution between the phenolic ring of Tyr² and the disulphide bridge Cys¹–Cys⁶. We also analyzed acrylamide quenching of tyrosyl fluorescence to determine the exposure of the tyrosyl ring to the solvent. Results from fluorescence experiments were compared with those from Monte Carlo simulation (ECEPP/3 force-field). Received: 5 August 1996 / Accepted: 8 December 1996     

Cys294 is essential for the function of the human sodium-dependent multivitamin transporter

The sodium-dependent multivitamin transporter (SMVT) plays an important role in biotin uptake in the intestine and other cell types. While significant knowledge has been gained with regard to regulation and cell biology of the SMVT system, there is little known about its structure-function relationships. Here we examined the role of each of the ten conserved (among species) cysteine residues in the function of the human SMVT (hSMVT) using site-directed mutagenesis. Our results showed a significant impairment in biotin uptake only in cells transfected with hSMVT mutated at Cys²⁹⁴, but not at the other conserved cysteine residues; the impairment in biotin uptake caused by mutating Cys²⁹⁴ was not related to the polar status of substituting amino acid. The inhibition in hSMVT function upon mutating Cys²⁹⁴ was mediated via a significant reduction in the V_max, but not the apparent K_m, of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not affinity. Biotinylation assay confirmed this suggestion by showing a marked reduction in the level of expression of the mutated protein at the cell membrane, without affecting total cellular level of induced hSMVT. These results show an important role for Cys²⁹⁴ in the function and cell biology of hSMVT.     

NMR structural elucidation of myelin basic protein epitope 83–99 implicated in multiple sclerosis

Myelin basic protein peptide 83–99 (MBP83–99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP_83–99 has been performed using 2D ¹H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined α-helix spanning residues Val⁸⁷–Phe⁹⁰ is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.     

Structural properties of the peroxiredoxin AhpC2 from the hyperthermophilic eubacterium Aquifex aeolicus

Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H₂O₂), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H₂O₂ in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8?Å resolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys⁴⁹, Cys²¹², and Cys²¹⁸. The peroxidatic cysteine C_P⁴⁹ was found to be hyperoxidized to the sulfonic acid (SO₃H) form, while Cys²¹² forms an intra-monomer disulfide bond with Cys²¹⁸. Mutagenesis experiments indicate that Cys²¹² and Cys²¹⁸ play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.