Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G→A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

X-ray Structure of Gelonin at 1.8 Å Resolution

Gelonin is a single chain ribosome inactivating protein (RIP) with potential application in the treatment of cancer and AIDS. Diffraction quality crystals grown using PEG3350, belong to the space group P2(1), with it a = 49.4 Angstrom b = 44.9 Angstrom, c = 137.4 Angstrom and beta = 98.4 degrees, and contain two molecules in the asymmetric unit. Diffraction data collected to 1.8 Angstrom resolution has a R(m) value of 7.3%. Structure of gelonin has been solved by the molecular replacement method, using ricin A chain as the search model. Crystallographic refinement using X-PLOR resulted in a model for which the r.m.s deviations from ideal bond lengths and bond angles are 0.012 Angstrom and 2.7 degrees, respectively The final R-factor is 18.4% for 39,806 reflections for which I > 1.0 sigma(I).The C-alpha atoms of the two molecules in the asymmetric unit superpose to within 0.38 Angstrom for 247 atom pairs. The overall fold of gelonin is similar to that of other RIPs such as ricin A chain and alpha-momorcharin, the r.m.s.d. for C-alpha superpositions being 1.3 and 1.4 Angstrom, respectively The-catalytic residues (Glu166, Arg169 and Tyr113) in the active site form a hydrogen bond scheme similar to that observed in other RIPs. The conformation of Tyr74 in the active site, however, is significantly different from that in alpha-momorcharin. Three well defined water molecules are located in the active site cavity and one of them, X319, superposes to within 0.2 Angstrom of a corresponding water molecule in the structure of alpha-momorcharin. Any of the three could be the substrate water molecule in the hydrolysis reaction catalysed by gelonin.Difference electron density for a N-linked sugar moiety has been observed near only one of the two potential glycosylation sites in the sequence. The amino acid at position 239 has been established as Lys by calculation of omit electron density maps.The two cysteine residues in the sequence, Cys44 and Cys50, form a disulphide bond, and are therefore not available for disulphide conjugation with antibodies. Based on the structure, the region of the molecule that is involved in intradimer interactions is suggested to be suitable for introducing a Cys residue for purposes of conjugation with an antibody to produce useful immunotoxins.

Heme biosynthesis by the malarial parasite - Import of delta-aminolevulinate dehydrase from the host red cell

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment, The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P, berghei infected mouse red cell in vivo, The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant, The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite, While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme, However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation, ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

Homology between jacalin and artocarpin from jackfruit (Artocarpus integrifolia) seeds. Partial sequence and preliminary crystallographic studies of artocarpin

Jacalin and artocarpin, the two lectins from jackfruit (Artocarpus integrifolia) seeds, have different physicochemical properties and carbohydrate-binding specificities. However, comparison of the partial amino-acid sequence of artocarpin with the known sequence of jacalin indicates close to 50% sequence identity. Artocarpin crystallizes in two forms, both monoclinic P2(1), with one and two tetramic molecules, respectively, in the asymmetric units of form I (a = 69.9, b = 73.7, c = 60.6 Angstrom and beta = 95.1 degrees) and form II (a = 87.6, b = 72.2, c = 92.6 Angstrom and beta = 101.1 degrees). Both the crystal structures have been solved by the molecular replacement method using the known structure of jacalin as the search model and ope of them partially refined, confirming that the two lectins are indeed homologous.

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

A 3-hour quantitative comparison of glucose-based versus rice-based oral rehydration solution intake by children with diarrhoea in Port Moresby General Hospital

Measurements were made of the intake of a WHO/UNICEF glucose-based and a rice cereal-based oral rehydration solution (ORS) by children with diarrhoea. Twenty children who presented to the Children's Outpatient Department at Port Moresby General Hospital with acute diarrhoea and mild dehydration were randomly assigned to an ORS and measurements were taken over the following 3 hours. For data analysis, the patients were paired by weight. Testing the means of the paired samples by t test showed that there was no significant difference between the amount of rice ORS and the amount of glucose ORS taken over 3 hours. The discovery of oral rehydration solution (ORS) for the treatment of diarrheal disease has been heralded as the most important medical discovery of the century. Cereal-based ORS is able to decrease stool output and the duration of diarrheal illness more than the standard glucose-based ORS, through the increased absorption provided by oligosaccharides without the imposition of a greater osmotic penalty. Moreover, the peptides in cereals enhance amino acid and water absorption, while providing nutritional benefits. UNICEF's glucose-based ORS is becoming more widely used in Papua New Guinea (PNG). 20 children aged 6-37 months (mean age, 15 months) who presented to the Children's Outpatient Department at Port Moresby General Hospital during September-October 1993 with acute diarrhea and mild dehydration were randomly assigned to receive either a rice-based ORS or standard glucose ORS, and measurements were taken over the following 3 hours. The patients were paired by weight for analysis. No statistically significant difference was found between the amount of rice ORS and the amount of glucose ORS taken over 3 hours.

Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-alpha to the receptor binding region of the beta-subunit

A single-step solid-phase RIA (SS-SPRIA) developed in our laboratory using hybridoma culture supernatants has been utilised for the quantitation of epitope-paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin (hCG) are differentially stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope at/near the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot region (C34-C38-C88-C90-H106). The carboxy terminal region of the alpha-subunit forms a part of the epitope indicating its proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified through other approaches and the X-ray crystal structure of hCG.

Comparative in vitro and in vivo studies of enteric-coated pancrelipase preparations for pancreatic insufficiency

We evaluated three acid-resistant pancreatic enzyme preparations by in vitro assays, and by comparing degree of steatorrhea, creatorrhea, fecal wet weight, and stool energy losses in a randomized crossover study of patients with pancreatic insufficient cystic fibrosis. Aims of the study were to assess (a) the most practicable and reliable indicator of malabsorption; (b) the variation in enzyme batch potency; (c) the decline in enzyme batch potency with prolonged shelf life; and (d) the relative bio-efficacy of the different preparations. In the in vivo study, absorption of energy, nitrogen, and fat did not differ when comparing the three preparations at roughly pharmaceu-tically equivalent doses, but when expressed per capsule of pancreatic supplement ingested, absorption reflected relative enzyme content, favoring the higher potency preparations. Although steatorrhea was reasonably controlled by these preparations, stool energy losses varied from 800 to 1,100 kJ per day, suggesting greater attention be paid to overall energy absorption rather than absorption of individual nutrients. In addition, fecal energy loss correlated more closely with fecal wet weight (r = 0.81; p < 0.05) than with steatorrhea (r = 0.40; ns), such that 1 g wet feces = 8.37 kJ (± 0.14). In vitro enzyme potency varied markedly between batches of the same brand, and also a decline of up to 20% in amylase, lipase, and trypsin activity was noted over an 8-month period for each batch. Both observations have clinical implications at times of represcription. Finally, the higher potency preparations were more effective per capsule and reduced capsule dosage is therefore attainable. © 1993 Raven Press, Ltd., New York.

Synthesis, crystal structures, DNA binding and cleavage activity of L-glutamine copper(II) complexes of heterocyclic bases

Ternary L-glutamine (L-gln) copper(II) complexes [Cu(L-gln)(B)(H2O)](X) (B = 2,2'-bipyridine (bpy), X = 0.5SO(4)(2-), 1; B = 1,10-phenanthroline (phen), X = ClO4-, 2) and [Cu(L-gln)(dpq)(ClO4)] (3) (dpq, dipyridoquinoxaline) are prepared and characterized by physicochemical methods. The DNA binding and cleavage activity of the complexes have been studied. Complexes 1-3 are structurally characterized by X-ray crystallography. The complexes show distorted square pyramidal (4+1) CuN3O2 coordination geometry in which the N,O-donor amino acid and the N, N-donor heterocyclic base bind at the basal plane with a H2O or perchlorate as the axial ligand. The crystal structures of the complexes exhibit chemically significant hydrogen bonding interactions besides showing coordination polymer formation. The complexes display a d-d electronic band in the range of 610-630 nm in aqueous-dimethylformamide (DMF) solution (9:1 v/v). The quasireversible cyclic voltammetric response observed near -0.1 V versus SCE in DMF-TBAP is assignable to the Cu(II)/Cu(I) couple. The binding affinity of the complexes to calf thymus (CT) DNA follows the order: 3 (dpq) > 2 (phen) >> 1 (bpy). Complexes 2 and 3 show DNA cleavage activity in dark in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent via a mechanistic pathway forming hydroxyl radical as the reactive species. The dpq complex 3 shows efficient photoinduced DNA cleavage activity on irradiation with a monochromatic UV light of 365 nm in absence of any external reagent. The cleavage efficiency of the DNA minor groove binding complexes follows the order:3 > 2 >> 1. The dpq complex exhibits photocleavage of DNA on irradiation with visible light of 647.1 nm. Mechanistic data on the photo-induced DNA cleavage reactions reveal the involvement of singlet oxygen (O-1(2)) as the reactive species in a type-II pathway. (C) 2008 Elsevier B.V. All rights reserved.

B- and T-cell epitopes of tropomyosin, the major shrimp allergen

Seafood allergy is often encountered on ingestion of crustaceans such as shrimp, lobster, crab, and crayfish (1). On eating cooked shrimp, sensitive individuals experience a wide spectrum of reactions ranging from abdominal discomfort to anaphylaxis. The presence of cross-reacting heat-stable allergens in crustacean food was first recognized by Hoffman et al. (2) and Lehrer et al. (3). Subsequently, the major allergen was isolated and characterized from the shrimp species Paneaus indicus (Pen i 1) (4) and I? aztecm (Pen a 1) (5). Pen i 1 (originally designated Sa-TI) and Pen a 1, with mol. mass of 34 and 36 kDa, respectively, contain 301 and 312 amino-acid residues with a predominance of gluta- mate/glutamine and asparatate/asparagine.