Half-cystinyl Residues Research Articles

Structure−Activity Relation of Human β-Defensin 3: Influence of Disulfide Bonds and Cysteine Substitution on Antimicrobial Activity and Cytotoxicity

Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.

Characteristics and primary structure of a galectin in the skin mucus of the Japanese eel, Anguilla japonica

The characteristics and primary structure of AJL-1, one of the lectins in the skin mucus of the Japanese eel ( Anguilla japonica), were examined. This lectin exhibited β-galactoside specific activity in a Ca 2+ independent manner. We previously reported that its molecular mass was 16,091 Da, although it was approximately 30 kDa as determined by gel filtration, indicating that it is a homodimer having non-covalent bonds. This lectin was composed of 142 amino acid residues having no half-cystinyl residues, and showed homology to members of the galectin family, especially to proto-type galectins. Gene expression of this lectin was detected in skin only, and relative expression was high in an individual that exhibited resistance to infectious disease. AJL-1 showed agglutinating activity against pathogenic bacteria, Streptococcus difficile. This suggests that AJL-1 functions as an important defensive factor at the body surface.

A novel gene overexpressed in the prostate of castrated rats: hormonal regulation, relationship to apoptosis and to acquired prostatic cell androgen independence.

We have identified a novel complementary DNA (cDNA) corresponding to a gene overexpressed in the rat ventral prostate after castration. This cDNA displays 89.4% identity with 453 bp of a mouse EST and 81.5% identity with 157 bp of a human EST and was named PARM-1 for prostatic androgen-repressed message-1. The complete cDNA is 1187 bp long and codes for a protein of 298 amino acids that contains four potential glycosylation sites and three half cystinyl residues. The PARM-1 gene was found to be expressed at quite low levels in most rat tissues including those of the urogenital tract. The kinetic of induction of PARM-1 gene in the prostate was highly correlated to the development of apoptosis in the whole organ. Supplementation of castrated animals with androgens reversed both the process of apoptosis and the overexpression of PARM-1 gene. Supplementation with estrogens did not result in an increase in the PARM-1 messenger RNA levels when compared with the castration alone. However, the treatment resulted in a more rapid return to intact levels in the castrated plus estrogen group. When apoptosis of testis and prostate was induced in vivo by hypophysectomy, it was found that PARM-1 was only overexpressed in the prostate. Therefore, PARM-1 seems to be regulated by androgens only in the prostate. Using in situ hybridization and immunohistological techniques, we have shown that PARM-1 gene product is found exclusively in the epithelial cells of involuting prostate. Analysis by flow cytometry of MAT LyLu epithelial cells transiently expressing PARM-1 protein did not allow us to demonstrate a direct effect of PARM-1 gene overexpression on the programmed death of the transfected cells. Treatment of MAT LyLu cells by transforming growth factor-beta induced apoptosis but had no effect on PARM-1 production. However PARM-1 protein has been detected by Western blotting in various cell lines such as MAT LyLu, MAT Lu, and PIF, which are androgen independent. This would suggest that PARM-1 gene product would be a marker for acquired androgen-independence of these tumor cells.

Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry

A new method is described for locating disulfide bonds in proteins which cannot be cleaved between half-cystinyl residues by enzymic methods, as is often the case for tightly coiled proteins, or for proteins in which half-cystinyl residues are not separated by residues required for enzymic cleavage. Partial acid hydrolysis of a model protein, hen egg-white lysozyme, produces a mixture of disulfide-containing peptides from which the disulfide connections may be deduced. The usefulness of a combination of HPLC, fast atom bombardment mass spectrometry, and computer-assisted analysis to identify disulfide-containing peptides present in the partial acid hydrolysate of the model protein is demonstrated. Chromatographic fractions of the hydrolysate were analyzed by mass spectrometry before and after chemical reduction of the disulfide bonds to determine the molecular weights of disulfide-containing peptides. Computer-assisted analysis was then used to relate the molecular weights of these peptides to specific segments of the protein from which the disulfide connectivities could be determined. Partial acid hydrolysis of proteins, which is attractive because it proceeds relatively independent of the amino acid sequence and structure, and because disulfide interchange is unlikely to occur in dilute acid, has become practical because disulfide-containing peptides present in complex mixtures can be identified rapidly and definitively by this method.

Isolation and properties of four serine protease inhibitors in the sea anemone Actinia equina.

Four serine protease inhibitors (AEPI-I, II, III and IV) were isolated from the sea anemone Actinia equina by acetone precipitation, gel filtration, ion-exchange HPLC and reversed-phase HPLC. They were all basic polypeptides with a molecular weight ranging from 6200 to 7000. The amino acid compositions of the inhibitors were very close to one another; they were characterized by the presence of 6 half-cystinyl residues but the absence of methionine and tryptophan. The inhibitors were active against serine proteases; inhibition for trypsin and α-chymotrypsin was displayed by the four inhibitors while that for kallikrein only by AEPI-I and II and that for plasmin only by AEPI-III and IV. However, none of the inhibitors showed affinity for both metalloproteases and cysteine proteases. These results suggested that the protease inhibitors can be classified into Kunitz-type inhibitors.

Complete amino acid sequence of the collagenase from the insect Hypoderma lineatum.

The primary structure of the Hypoderma lineatum collagenase was determined. Chymotrypsin digestion and thermolysin fragmentation of the chymotryptic core gave 30 and 5 peptides, respectively, accounting for all the residues of the protein. These peptides were aligned with overlapping peptides derived from tryptic and Staphylococcus aureus V8 proteinase digests. Hypoderma collagenase is a serine proteinase composed of 230 amino acids (Mr 25,223). It displays a high degree of sequential homology with the serine proteinases of the trypsin family, especially with another collagenolytic enzyme, the proteinase I of the crab Uca pugilator. The six half-cystinyl residues of Hypoderma collagenase correspond to 6 of the 10 half-cystinyl residues of chymotrypsin, and the residues forming the charge-relay system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions. The prediction of the secondary structure of the collagenase is given.

Amino acid sequence of human von Willebrand factor.

The complete amino acid sequence of human von Willebrand factor (vWF) is presented. Most of the sequence was determined by analysis of the S-carboxymethylated protein. Some overlaps not provided by the protein sequence analysis were obtained from the sequence predicted by the nucleotide sequence of a cDNA clone [Sadler, J.E., Shelton-Inloes, B.B., Sorace, J., Harlan, M., Titani, K., & Davie, E.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6391-6398]. The protein is composed of 2050 amino acid residues containing 12 Asn-linked and 10 Thr/Ser-linked oligosaccharide chains. One of the carbohydrate chains is linked to an Asn residue in the sequence Asn-Ser-Cys rather than the usual Asn-X-Ser/Thr sequence. The sequence of von Willebrand factor includes several regions bearing evidence of internal gene duplication of ancestral sequences. The protein also contains the tetrapeptide sequence Arg-Gly-Asp-Ser (at residues 1744-1747), which may be a cell attachment site, as in fibronectin. The amino- and carboxyl-terminal regions of the molecule contain clusters of half-cystinyl residues. The sequence is unique except for some homology to human complement factor B.

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues.

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins.

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.

Amino acid sequence of small and large subunits of seed storage protein from Ricinus communis.

The low molecular weight, glutamine-rich storage protein isolated from the seeds of Ricinus communis (castor beans) has been shown to consist of two different polypeptide chains linked by disulfide bond(s). The small subunit is composed of 34 amino acids with a proline at its NH2 terminus, whereas the large subunit contains 61 amino acids with a cyclized glutamine as the NH2-terminal residue. The complete amino acid sequence of both subunits has been determined through characterization of the isolated subunits and selected peptides from trypsin, chymotrypsin, thermolysin, and cyanogen bromide cleavage. The intact protein possesses a large number of glutaminyl and half-cystinyl residues and exhibits sequence heterogeneity as observed from peptide sequences. Comparison of the sequence of this protein and those of other seed proteins indicates some structural similarities between them. The amino acid sequences of the two polypeptide chains of castor bean storage protein are: (formula, see text).

Metallothionein occurs in roots of Agrostis tolerant to excess copper

Metallothioneins have an important function in metal metabolism in animals1. These low molecular weight proteins have high contents of half-cystinyl residues (up to 33%) which can bind metals such as Zn, Cu, Cd, Hg or Ag in mercaptide complexes1. In normal conditions animal tissues have low concentrations of metallothioneins, but these increase on administration of metal2–4, a reaction which is consistent with their suggested role in detoxification mechanisms2,5,6. Various plant genotypes exhibit tolerance to excesses of metals7, and Jowett8 proposed that such genotypes contain compounds capable of specific metal chelation. Metallothioneins could serve such a role. We report here that copper-thionein occurs in the roots of a clone of Agrostis gigantea Roth tolerant to excess copper9. This is the first unequivocal demonstration of a metallothionein in a vascular plant. These unusual proteins may have a role in metal metabolism in plants and could be important in the elucidation of the mechanism of metal tolerance.

Amino acid sequence of a collagenolytic protease from the hepatopancreas of the fiddler crab, Uca pugilator.

The amino acid sequence of a collagenolytic protease from the hepatopancreas of the fiddler crab, Uca pugilator, was determined from the structures of overlapping tryptic, chymotryptic, thermolytic, staphylococcal protease, and cyanogen bromide peptides together with automated sequencer analysis of the intact protein. Crab collagenase is a serine protease composed of 226 residues which is capable of degrading the native triple helix of collagen under physiological conditions. When aligned for optimal homology, crab collagenase displays 35% identity with bovine trypsin, 38% with bovine chymotrypsin B, and 32% with porcine elastase. The six half-cystinyl residues in crab collagenase correspond to those forming three of the five disulfide bonds in chymotrypsin. The residues forming the charge relay system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions in crab collagenase, and the sequences around these residues are well conserved. The primary structure of crab collagenase is the first reported for a serine protease from crustacean hepatopancreas and the first reported for a serine protease possessing the unusual property of being able to degrade native helical collagen.

D-3-Phosphoglycerate dehydrogenase from chicken liver. II. Chemical and physical properties.

The molecular weight of chicken liver D-3-phosphglycerate dehydrogenase, as measured by sedimentation equilibrium analysis, was found to be 165,000, consistent with the measured sedimentation coefficient of 8.13 S. Sedimentation equilibrium studies in 6 M guanidine hydrochloride and gel electrophoresis in sodium dodecyl sulfate gave molecular weights of 40,000 to 43,000, indicating that the protein is composed of 4 subunits of equal molecular weight. Fractionation of the soluble tryptic peptides of the S-[14C]carboxymethylated enzyme by ion exchange chromatography and unidirectional paper electrophoresis indicated a maximum of 40 major peptides, which is approximately one-fourth of the number of tryptic peptides expected from the lysine plus arginine content of 157 of the tetramer. This observation suggests that the subunits possess identical or very similar amino acid sequences. The purified enzyme is a basic protein with an isoelectric point of 8.95. Amino acid analysis and titration with p-hydroxymercuribenzoate indicated that the 12 half-cystinyl residues present/subunit exist in the free sulfhydryl form. NH2-terminal analysis in an automatic protein sequenator indicated that the polypeptide chains of the protein are blocked.

Snake Venom Proteinase Inhibitors: III. Isolation of Five Polypeptide Inhibitors from the Venoms of Hcmachatus haemachatus (Ringhal's Cobra) and Naja nivea (Cape Cobra) and the Complete Amino Acid Sequences of Two of Them1

Five proteinase inhibitors which all inhibit the activity of bovine trypsin [EC 3.4.21.4] were isolated from African Elapid venoms of Hemachatus haemachatus (HHV, Ringhal's cobra) and Naja nivea (NNV, Cape cobra). All the inhibitors were essentially homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecylsulfate. Amino acid analysis and terminal analysis also supported their chemical homogeneities, except for one of the two inhibitors from Hemachatus haemachatus venom. The isolated inhibitors had a molecular weight of about 6,500, consisting of 52 to 57 amino acid residues, and they were all devoid of tryptophan. However, their amino acid compositions differed from each other. One of the three inhibitors isolated from Naja nivea venom, designated NNV inhibitor Ia, was unique, in that 4 half-cystinyl residues per mole fof the polypeptide were present, whereas all the others contained six residues. Of the isolated proteinase inhibitors, the complete amino acid sequences of two major inhibitors were established by manual and automatic Edman degradations and standard enzymatic techniques. Each of the inhibitors, designated HHV inhibitor II and NNV inhibitor II, consisted of 57 amino acid with arginine and glycine at the NH2- and COOH-termini, respectively. Both contained six half-cystines in disulfide linkages, and their overall amino acid sequences were similar, showing 91% homology. The two inhibitors differed in sequence by only five amino acid replacements, Asp-3 to Arg; Tyr-17 to Arg; Leu-25 to Arg; Gln-32 to Glu; and Arg-52 to His, in the 57 residue peptide chain. Comparing the amino acid sequences of these two cobra venom inhibitors with those of Russell's viper venom inhibitor II and bovine pancreatic trypsin inhibitor (BPTI), about 50% homology was found in their sequences. The 6 half-cystinyl residues of these inhibitors were in the same linear positions. Moreover, the regions which are structurally and functionally important in the well-known BPTI molecule were found with extremely high sequence homology in the cobra venom inhibitors. These findings strongly suggest that the cobra venom inhibitors as well as Russell's viper inhibitor II have very similar conformations to that established for BPTI.

Nerve growth factor: structure and mechanism of action.

This chapter presents the structure and mechanism of action of nerve growth factor (NGF). NGF has been purified to homogeneity from the submaxillary gland of the adult male mouse and from the venom of the cobra Naja naja . Both proteins are dimers of identical polypeptide chains which have a molecular weight of about 13,000. The chapter presents the primary structures of mouse and cobra NGF. The mouse NGF sequence is the continuous line, and the different residues that appear in the tentative Naja naja NGF sequence are indicated at their respective positions below the mouse sequence. The polypeptide chain of mouse NGF is 118 residues while that of cobra NGF is 117 residues. Three intramolecular disulfide bridges link half-cystinyl residues 15 to 80, 58 to 108, and 68 to 110 in the mouse protein and, as judged by the identical location of the half-cystinyl residues, in the Naja naja protein as well. The proteins from these two sources are quite similar in amino acid sequence, having identical amino acid residues in about 65% of the positions in their sequences.

Nerve growth factor

Publisher Summary This chapter discusses the structure of nerve growth factor (NGF). The amino acid sequence of mouse NGF exhibited significant structural relatedness with the family of insulins and proinsulins. The sequence of mouse NGF could be aligned with human proinsulin with only five deletions required to yield the maximum homology of 21% identical residue positions. This is indicative of distant, but significant evolutionary relatedness. The majority of the identical residues were seen to be clustered in the segments of NGF, which aligned with the insulin A and B chain regions of the proinsulin sequence, separated by exactly the 35 residues required to accommodate the C activation peptide of proinsulin. Thus, the order of similar regions of NGF is B chain-C peptide-A chain, the same order in which they occur in proinsulin. Because NGF is 118 residues and human proinsulin is 86 residues, 37 residues of the C terminal region of NGF extend beyond the proinsulin molecule. This region is, however, similar to insulin B chain, suggesting a repetitive structure arising from a gene duplication event. It is also noteworthy that three out of the six half cystinyl residues that occur in both proteins are in identical positions, and two of these remain paired in identical fashion in insulin and NGF.

Characterization of a photoexposed sulfhydryl group of bovine rhodopsin available for chemical modification

Summary Alkylation of rhodopsin in 40 mM cetyltrimethylammonium bromide revealed that the ratio of the light:dark formation of [ 14 C]carbamidomethylated-opsin was 1.0:0.1. A rhodopsin photo-exposed sulfhydryl group was demonstrated in rod outer segment membranes with [ 14 C]parachloromercuribenzoate and [ 14 C]iodoacetamide. Amino acid analysis of native and S-carbamidomethylcysteinyl-opsin, before and after performic acid oxidation, indicated that 1.2–1.3 sulfhydryl groups were available for alkylation per 4.9 total half-cystinyl residues in illuminated rhodopsin.

L-3-Hydroxyacyl Coenyzme A Dehydrogenase from Pig Heart Muscle: I. PURIFICATION AND PROPERTIES

Abstract A method is described for the preparation of homogeneous 3-hydroxyacyl-CoA dehydrogenase from pig heart muscle in amounts sufficient for structural studies. The procedure utilizes ammonium sulfate precipitation, gel filtration, and ion exchange chromatography on CM-cellulose. The homogeneity of the preparation obtained has been established by isoelectric focusing, sedimentation analysis, starch gel electrophoresis, and sodium dodecyl sulfate gel electrophoresis. Crystals of the protein have been obtained with ammonium sulfate in phosphate buffer at pH 6.8. The enzyme exhibits marked substrate inhibition with acetoacetyl-CoA, and to a lesser extent with S-acetoacetyl pantetheine, when assayed in the direction of NADH oxidation. The activity with S-acetoacetyl-N-acetylcysteamine is too low to determine whether high levels of this substrate also inhibit the enzyme. The purified enzyme is a basic protein with an isoelectric point of 8.95 at 4°. It possesses a sedimentation coefficient of 3.88 S and a maximum ultraviolet absorption at 278 nm. The extinction coefficient at this wave length is E1%1 cm = 4.39. The amino acid composition of the enzyme has been determined. The most notable features are 2 residues of tryptophan and 2 residues of half-cystine per mole of protein. Both half-cystinyl residues are present in the native enzyme as cysteine as judged by titration of the protein in 6 m guanidine hydrochloride with p-chloromercuribenzoate.

The Primary Structure of Epidermal Growth Factor

The complete amino acid sequence of epidermal growth factor (EGF) has been established through the use of automated Edman degradation and standard enzymatic and chemical techniques. The location of the half-cystinyl residues was facilitated by the use of the S-[14C]aminoethyl derivative of EGF. The sequence is: [see PDF for sequence]. The calculated molecular weight of the 53-residue polypeptide is 6045, a value that is in agreement with the molecular weight of 6400 established by physical measurements. The peptide is acidic and the 6 half-cystines exist in disulfide linkage. Four arginine residues are located in the COOH-terminal portion of the molecule. The six COOH-terminal amino acids are not necessary for biological activity and EGF1–47 has activity identical with that of the 53-residue native molecule.

Nerve growth factor from mouse submaxillary gland: amino acid sequence.

The complete amino-acid sequence of the 2.5S nerve growth factor from male-mouse submaxillary glands has been determined. The unambiguous alignment of peptides derived from tryptic, chymotryptic, thermolytic, and peptic digestion of S-carboxymethyl-, S-aminoethyl-, and native growth factor indicates that the primary subunit is composed of 118 amino acids, with amino-terminal serine and carboxyl-terminal arginine. The molecular weight of this subunit, calculated from the primary sequence, is 13,259. Thus, the native protein, which is composed of two of the subunits, has a molecular weight of 26,518. These values, as well as the final amino-acid composition, are in excellent agreement with those determined by direct measurement with undigested growth factor. The alignment of the three disulfide bonds, determined from a combination of peptic and thermolytic digestions, is I-IV, II-V, and III-VI. The latter two pairs are located in a closed loop of 14 amino acids, by virtue of the fact that half-cystinyl residues V and VI are separated by only a single residue in the linear sequence. Assignment of the side-chain amides showed that 7 of 11 aspartic acid residues and 2 of 8 glutamic acid residues are present as amides. This distribution of charged residues is entirely consistent with the observed isoelectric point of 9.3.