Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin

Antibacterial peptides were isolated from porcine spleen by acetic acid extraction, ion exchange chromatography and reverse-phase high-performance liquid chromatography. C-terminal ladder sequence analysis of a bioactive peptide with matrix-assisted laser desorption/ionization mass spectrometry after digestion with carboxypeptidases P and Y showed that it is identical to the antibacterial proline/arginine-rich intestinal peptide PR-39. It is present at high levels in granulocytes of the spleen, and peptides with C-terminal proline amide and internal adjacent Pro residues can be analyzed with this method. In addition, two forms of NK-lysin (NKL) were found. One, NKLi, is identical to that isolated from pig intestine, and the other, NKLbw, to a mature peptide deduced from a clone from a porcine bone marrow cDNA library.     

Trefoil factors

The present review will include the mammalian trefoil factors, TFF1, TFF2 and TFF3. It will summarise the amino acid sequences from different species, their posttranslational modifications and their structures determined by X-ray analysis and nuclear magnetic resonance studies. Trefoil factors all have a well-defined, structurally conserved trefoil domain. The trefoil domain consists of 42 or 43 amino acid residues and contains 6 cysteine residues that form disulphide bonds in a 1-5, 2-4 and 3-6 configuration. By the establishment of an additional intra-molecular disulphide bond at the C-terminal end, TFF1 and TFF3 form homodimers or heterodimers. This dimer formation of TFF1 and TFF3 will be discussed, and the possible implications for biological activity will be reviewed. The physicochemical characteristics including protease stability of trefoil factors will be summarised. The biological implications of different molecular forms of trefoil factors and their interaction with mucins will be discussed together with other functional insights.     

Hypotensive activity of histidine-containing analogues of C-terminal hexapeptide of substance P

Four new hexapeptide analogues of C-terminal Substance P fragment with increased solubility in aqueous solutions are described. The peptides contain histidine in positions 6, 8, 9 and 10, respectively. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of amino acid residues in various positions in the C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive and antigenic properties, respectively. Only the [His6] SP6-11 analogue had an unchanged antigenic structure when compared with the C-terminal region of Substance P, but it showed an almost total loss of hypotensive activity. The [His9] SP6-11 analogue retained 50% of the hypotensive activity of the C-terminal hexapeptide but showed a markedly reduced expression of the antigenic epitope localized in this region of Substance P.     

CD23 (the low-affinity IgE receptor) as a C-type lectin: a multidomain and multifunctional molecule

This review, regards the low-affinity receptor CD23 as a C-type lectin and compares it with other C-type lectins and C-type lectin-like receptors. C-type lectins such as the asialoglycoprotein receptor, as well as the dendritic cell immunoreceptor and the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin on dendritic cell lectin, possess amino acid sequences which interact with Ca⁺⁺ and sugar, and many of them possess an endocytosis signal sequence that includes tyrosine or serine in the cytoplasmic region. In contrast, natural killer receptors lack the Ca⁺⁺ and sugar-binding amino acids but conserve homologous cysteines in the form of C-type lectin, and possess an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic region which inhibits killer activity when they recognize the self major histocompatibility (MHC) class I molecule. Since human CD23a form has a similar amino acid sequence, the possibility that this sequence is an endocytosis signal or an ITIM is discussed. The function of the reverse RGD and RGD-binding inhibitory peptide in human CD23 from the point of view of the relation between a C-type lectin and MHC class II molecules is also considered. Received 21 May 2001; received after revision 28 November 2001; accepted 29 November 2001     

Hypotensive activity of histidine-containing analogues of C-terminal hexapeptide of Substance P

Summary Four new hexapeptide analogues of C-terminal Substance P fragment with increased solubility in aqueous solutions are described. The peptides contain histidine in positions 6, 8, 9 and 10, respectively. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of amino acid residues in various positions in the C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive and antigenic properties, respectively. Only the [His⁶] SP_6-11 analogue had an unchanged antigenic structure when compared with the C-terminal region of Substance P, but it showed an almost total loss of hypotensive activity. The [His⁹] SP_6-11 analogue retained 50% of the hypotensive activity of the C-terminal hexapeptide but showed a markedly reduced expression of the antigenic epitope localized in this region of Substance P.     

Lactoferrin

Lactoferrin (Lf), a prominent protein in milk, many other secretory fluids and white blood cells, is a monomeric, 80-kDa glycoprotein, with a single polypeptide chain of about 690 amino acid residues. Amino acid sequence relationships place it in the wider transferrin family. Crystallographic analyses of human Lf, and of the Lfs from cow, horse, buffalo and camel, reveal a highly conserved three-dimensional structure, but with differences in detail between species. The molecule is folded into homologous N- and C-terminal lobes, each comprising two domains that enclose a conserved iron binding site. Iron binding and release is accompanied by domain movements that close or open the sites, and is influenced by cooperative interactions between the lobes. Patches of high positive charge on the surface contribute to other binding properties, but the attached glycan chains appear to have little impact on structure and function.     

Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei

Three novel glycine-rich peptides, named ctenidin 1–3, with activity against the Gram-negative bacterium E. coli, were isolated and characterized from hemocytes of the spider Cupiennius salei. Ctenidins have a high glycine content (>70%), similarly to other glycine-rich peptides, the acanthoscurrins, from another spider, Acanthoscurria gomesiana. A combination of mass spectrometry, Edman degradation, and cDNA cloning revealed the presence of three isoforms of ctenidin, at least two of them originating from simple, intronless genes. The full-length sequences of the ctenidins consist of a 19 amino acid residues signal peptide followed by the mature peptides of 109, 119, or 120 amino acid residues. The mature peptides are post-translationally modified by the cleavage of one or two C-terminal cationic amino acid residue(s) and amidation of the newly created mature C-terminus. Tissue expression analysis revealed that ctenidins are constitutively expressed in hemocytes and to a small extent also in the subesophageal nerve mass.     

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1–212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1–212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1–3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region. Received 24 April 2007; received after revision 11 June 2007; accepted 13 June 2007     

Chemical characterization of human urine albumin in proteinuria.

From the urine of a patient with proteinuria, the albumin protein component was isolated and compared with human serum albumin. By comparing the amino acid composition of the original proteins and their large cyanogen bromide fragments, peptide maps and N-terminal sequences of 33 amino acid residues, the identity of both proteins was shown.     

Chemical synthesis of a hexadecapeptide segment of ubiquitin that activates adenylate cyclase and induces lymphocytes to differentiate.

A hexadecapeptide corresponding to positions 59--74 of ubiquitin was synthesized and purified. The peptide was characterized by its mobility in TLC and electrophoresis, amino acid sequence and composition, and molar rotation. The peptide possessed approximately 40% activity compared with native ubiquitin in each of 3 biological assays in vitro: a) thymocyte induction, b) B cell induction and c) elevation of intracellular cyclic AMP levels in sarcoma 180 cells.     

A new solid-phase synthesis of porcine vasoactive intestinal peptide using Nα-9-fluorenylmethyloxycarbonyl amino acids

Summary The solid-phase synthesis of an octacosapeptide amide corresponding to the amino acid sequence of porcine vasoactive intestinal peptide (VIP) is described. No final treatment with strong, anhydrous acid was employed, since the use of base-labile 9-fluorenylmethyloxycarbonyl amino acids bearing tert-butyl based side chain protection enabled the peptide chain assembly to be performed on p-benzyloxybenzyl amine resin, which was cleaved from the whole peptide amide at the end of the synthesis by diluted trifluoroacetic acid.     

Proinsulin C-peptide and its C-terminal pentapeptide: degradation in human serum and Schiff base formation with subsequent CO<Subscript>2</Subscript> incorporation

Processing of human proinsulin C-peptide and its C-terminal pentapeptide in blood serum was studied using reverse-phase HPLC and electrospray mass spectrometry. The results reveal degradation of both peptides, with a longer half-life for intact C-peptide than for the C-terminal pentapeptide. Products from C-peptide degradation were not distinguishable from the peptide background, suggesting endopeptidase degradation of C-peptide. In contrast, a set of products from the C-terminal pentapeptide were identifiable and corresponded to successive losses from the N terminus, showing that the pentapeptide is degraded by aminopeptidase in serum. Consistent with this finding, a slower degradation was found for the N-acetyl-protected pentapeptide. Removal of serum proteins by acetone precipitation produced N-terminally carbamate-modified C-peptide via a Schiff base intermediate (a ketimine with acetone), to which CO(2) was added and acetone removed, generating a cyclic side chain via anhydride formation. The modification was not seen with the pyroglutamate form of C-peptide, with the N-terminally acetylated C-peptide, or with a control peptide having N-terminal Phe, but was found with human C-peptide, its N-terminal tetrapeptide, and a rat C-peptide fragment (all with N-terminal Glu). Hence, the modification appears to require N-terminal Glu, but this is not the only prerequisite since the C-terminal pentapeptide and another control peptide (also starting with Glu) were not modified. A peptide aldimine Schiff base leading to CO(2) incorporation was detected with formaldehyde in NaHCO(3). The observation that C-peptide forms Schiff bases with ketones/aldehydes, enhancing covalent attachment of CO(2), may have biological implications.     

Sterol carrier protein-2: structure reveals function

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.     

The C-terminal sequence of human and porcine antithrombin III and its homology with human α-1-proteinase inhibitor

Summary The C-terminal amino acid sequences of human and of porcine antithrombin III have been determined as Gly-Arg-Val-Ala-Asn-Pro-Cys-Val-Lys and Gly-Arg-Val-Ala-Asn-Pro-Cys, respectively. These sequences are highly homologous with the C-terminal sequence of human -1-proteinase inhibitor.     

Degradation of encephalitogenic protein in aerobic ascorbic acid solutions

Summary Aerobic ascorbic acid solutions are capable of extensively cleaving the peptide chain of the myelin basic protein. Cleavage occurred most readily with C-terminal to aspartic acid, serine, threonine, glutamic acid and leucine residues.This research was supported in part by the National Multiple Sclerosis Society, grant number RG928-A-1.     

Degradation of encephalitogenic protein in aerobic ascorbic acid solutions.

Aerobic ascorbic acid solutions are capable of extensively cleaving the peptide chain of the myelin basic protein. Cleavage occurred most readily with C-terminal to aspartic acid, serine, threonine, glutamic acid and leucine residues.     

The role of key residues in structure, function, and stability of cytochrome-c

Cytochrome-c (cyt-c), a multi-functional protein, plays a significant role in the electron transport chain, and thus is indispensable in the energy-production process. Besides being an important component in apoptosis, it detoxifies reactive oxygen species. Two hundred and eighty-five complete amino acid sequences of cyt-c from different species are known. Sequence analysis suggests that the number of amino acid residues in most mitochondrial cyts-c is in the range 104?±?10, and amino acid residues at only few positions are highly conserved throughout evolution. These highly conserved residues are Cys14, Cys17, His18, Gly29, Pro30, Gly41, Asn52, Trp59, Tyr67, Leu68, Pro71, Pro76, Thr78, Met80, and Phe82. These are also known as “key residues”, which contribute significantly to the structure, function, folding, and stability of cyt-c. The three-dimensional structure of cyt-c from ten eukaryotic species have been determined using X-ray diffraction studies. Structure analysis suggests that the tertiary structure of cyt-c is almost preserved along the evolutionary scale. Furthermore, residues of N/C-terminal helices Gly6, Phe10, Leu94, and Tyr97 interact with each other in a specific manner, forming an evolutionary conserved interface. To understand the role of evolutionary conserved residues on structure, stability, and function, numerous studies have been performed in which these residues were substituted with different amino acids. In these studies, structure deals with the effect of mutation on secondary and tertiary structure measured by spectroscopic techniques; stability deals with the effect of mutation on T _m (midpoint of heat denaturation), ?G _D (Gibbs free energy change on denaturation) and folding; and function deals with the effect of mutation on electron transport, apoptosis, cell growth, and protein expression. In this review, we have compiled all these studies at one place. This compilation will be useful to biochemists and biophysicists interested in understanding the importance of conservation of certain residues throughout the evolution in preserving the structure, function, and stability in proteins.     

Chemical synthesis of a hexadecapeptide segment of ubiquitin that activates adenylate cyclase and induces lymphocytes to differentiate

Summary A hexadecapeptide corresponding to positions 59–74 of ubiquitin was synthesized and purified. The peptide was characterized by its mobility in TLC and electrophoresis, amino acid sequence and composition, and molar rotation. The peptide possessed approximately 40% activity compared with native ubiquitin in each of 3 biological assays in vitro: a) thymocyte induction, b) B cell induction and c) elevation of intracellular cyclic AMP levels in sarcoma 180 cells.Acknowledgments. We thank Dr Geoffrey Tregear for helpful suggestions for the synthesis strategy, Dr Jim Burton for guidance with the methods for establishing peptide purity, and Ronald King, Miriam Miller, Jeanette Dilley and Linda Townley for excellent technical assistance. This work was supported by U.S. Public Health Service Grants CA-08748, CA-16889, CA-08415, CA-17085 and AI-12487, and by NCI Contract CB-53868.     

Isolation and characterization of hainantoxin-IV,a novel antagonist of tetrodotoxin-sensitive sodium channels from the Chinese bird spider <Emphasis Type="Italic">Selenocosmia hainana</Emphasis>

A neurotoxin, named hainantoxin-IV, was purified from the venom of the spider Selenocosmia hainana. The amino acid sequence was determined by Edman degradation, revealing it to be a 35-residue polypeptide amidated at its C terminal and including three disulfide bridges: Cys2-Cys17, Cys9-Cys24, and Cys16-Cys31 assigned by partial reduction and sequence analysis. Hainantoxin-IV shares 80% sequence identity with huwentoxin-IV from the spider S. huwena, a potent antagonist that acts at site 1 on tetrodotoxin-sensitive (TTX-S) sodium channels, suggesting that hainantoxin-IV adopts an inhibitor cystine knot structural motif like huwentoin-IV. Under whole-cell voltage-clamp conditions, this toxin has no effect on tetrodotoxin-resistant voltage-gated sodium channels in adult rat dorsal root ganglion neurons, while it blocks TTX-S sodium channels in a manner similar to huwentoxin-IV, and the actions of both toxins on sodium currents are very similar to that of tetrodotoxin. Thus, they define a new family of spider toxins affecting sodium channels.     

Structural differences between somatic H2B and testis-specific TH2B histones of the rat

Summary Testis-specific histone TH2B from rat testis showed a very similar tryptic peptide map to somatic H2B from rat liver, indicating a common evolutionary origin. However, 5 peptide differences were detected between the 2 proteins, and the amino acid compositions of these distinctive peptides were determined.Supported by Grant 780-0609 from the Ford Foundation.