Molecular relationship between two types of phospholipase B from Penicillium notatum and reconstitution of active enzyme from its peptide fragments

Two types of phospholipase B of Penicillium notatum, the native type and the modified type that is thought to be generated by the introduction of some nicks into the native type of enzyme by the endogenous protease(s), were distinguished on a slab sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under a nonreducing condition. The native form migrated with a rate corresponding to 95K Da, whereas the modified form migrated more slowly, corresponding to 106K Da, presumably because of its more extended conformation. That the "106K" protein was indeed a nicked product of the "95K" protein was confirmed by amino acid analysis, peptide mapping, N- and C-terminal sequence analyses, and immunoblotting. The peptide fragments (70K and 37K + 32K) comprising the modified protein were isolated by gel filtration in the presence of SDS and 2-mercaptoethanol (the 32K peptide was suggested to be a partial proteolytic product of the 37K peptide). When the "95K" protein was subjected to the same treatment under denaturing condition, it retained a low, but significant, enzymatic activity; in contrast, the separated peptide fragments did not show any significant activity. By a coincubation of these fragments, however, a restoration of enzymatic activity was observed through a reformation of the active complex, corresponding to the original modified protein. The enzymatic activity of this complex was further increased by a treatment with guanidine X HCl, followed by dialysis. The association of peptide fragments appears to occur through the formation of interpeptidal disulfide bonds.     

Identification of minimal peptide sequences in the (8-20) domain of human islet amyloid polypeptide involved in fibrillogenesis

We have examined a series of overlapping peptide fragments from the 8-20 region of human islet amyloid polypeptide (IAPP) with the objective of defining the smallest fibril-forming domain. Peptide fragments corresponding to LANFLV (residues 12-17) and FLVHSS (residues 15-20) were strong enhancers of beta-sheet transition and fibril formation. Negative stain electron microscopy illustrated the ability of these peptide fragments to form fibrils independently when incubated alone in solution. Circular dichroism analysis revealed that when full-length human IAPP was incubated in the presence of these two fragments, fibrillogenesis was accelerated. While the two fragments, LANFLV and FLVHSS, were able to enhance the recruitment of additional IAPP molecules during fibril formation, the "seeding" activity of these peptides had no effect on altering IAPP-induced cytotoxcity as determined by cell culture studies. Therefore, this study has identified two internal IAPP peptide fragments within the 8-20 domain that may have a role in enhancing the folding and aggregation of human IAPP. These fragments are the smallest sequences identified, within the 8-20 region of hIAPP, that can independently form fibrils, and that can interact with IAPP to assemble into fibrils with characteristics similar as those formed by human IAPP alone.     

Amino Acid Sequences of Neuropeptides in the Sinus Gland of the Land Crab Cardisoma carnifex: A Novel Neuropeptide Proteolysis Site

The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.     

Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin

Caldesmon is known to bind to smooth muscle myosin. Ca2+/calmodulin-dependent phosphorylation of caldesmon completely blocks its interaction with myosin. Cleavage of caldesmon at its 2 cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) occurs initially at one site to yield 108-kDa and 21.2-kDa peptides and subsequently at the second site within the 108-kDa peptide to yield 85-kDa and 23.5-kDa fragments. The 23.5-kDa peptide retains the ability to bind to myosin. The N-terminal (95 kDa) and C-terminal (42 kDa) chymotryptic peptides of caldesmon were isolated and digested with NTCB: the C-terminal actin- and calmodulin-binding peptide was not cleaved, indicating that it does not contain either of the cysteine residues, whereas the 95-kDa N-terminal peptide was cleaved at two sites to yield 56-kDa, 23.5-kDa, and 21.2-kDa fragments. The arrangement of NTCB fragments in caldesmon is, therefore: 21.2 kDa/23.5 kDa/85 kDa from N to C terminus. Digestion of phosphorylated caldesmon with NTCB suggested a single phosphorylation site in the 21.2-kDa peptide and three sites in the 23.5-kDa peptide. These results lead to the development of a model whereby caldesmon may cross-link actin to myosin and such cross-linking is blocked by phosphorylation of caldesmon. This mechanism may explain the formation of reversible "latch bridges" which permit force maintenance at low levels of myosin phosphorylation in intact smooth muscles.     

Isolation of a cDNA coding for human galactosyltransferase

Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a lambda gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 X 10(6) recombinant lambda gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.     

Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectrometric analysis after in gel chymotryptic digestion

To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis.     

Functionally important segments in proteins dissected using Gene Ontology and geometric clustering of peptide fragments

We have developed a geometric clustering algorithm using backbone ,ψ angles to group conformationally similar peptide fragments of any length. By labeling each fragment in the cluster with the level-specific Gene Ontology 'molecular function' term of its protein, we are able to compute statistics for molecular function-propensity and p-value of individual fragments in the cluster. Clustering-cum-statistical analysis for peptide fragments 8 residues in length and with only trans peptide bonds shows that molecular function propensities ≥20 and p-values ≤0.05 can dissect fragments within a protein linked to the molecular function.     

Sites of phosphorylation and mutation in regulatory subunit of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells: mapping to structural domains

A novel peptide mapping approach has been used to map sites of charge modification to major structural domains of regulatory subunit (R) of type I cAMP-dependent protein kinase from S49 mouse lymphoma cells. Proteolytic fragments of crude, radiolabeled R were purified by cAMP affinity chromatography and displayed by two-dimensional polyacrylamide gel electrophoresis. [35S]methionine-labeled peptides containing sites of mutation or phosphorylation exhibited charge heterogeneity attributable to the modification. Phosphate-containing fragments were also labeled with [32P]orthophosphate to confirm their phosphorylation. Major fragments from [35S]methionine-labeled S49 cell R corresponded in size to carboxyterminal cAMP-binding fragments reported from proteolysis of purified type I Rs from various mammalian species; additional fragments were also visualized. End-specific markers in Rs from some mutant S49 sublines confirmed that cAMP-binding fragments extended to the carboxyterminus of R. Aminoterminal endpoints of fragments could be deduced, therefore, from peptide molecular weights. Clustering of proteolytic cleavage sites within the "hinge-region" separating aminoterminal and carboxyterminal domains of R permitted high resolution mapping in this region: the endogenous phosphate and a "phenotypically-silent" electrophoretic marker mutation fell within a 2.5-kdalton interval at its aminoterminal end. On the other hand, Ka mutations that increase the apparent constant for activation of kinase by cAMP mapped within the large cAMP-binding region of R. A map of charge density distribution within the hinge-region of R was constructed to facilitate structural comparisons between Rs from S49 cells and from other mammalian sources.     

Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.     

Immunoreactivity of proteolytic degradation products of human prostatic acid phosphatase

Prostatic acid phosphatase (EC 3.1.3.2) was fragmented by trypsin and papain in the presence of sodium dodecyl sulphate. Trypsin-catalysed cleavage gave a peptide of 33 kDa which was subsequently trimmed to 18 kDa, 15 kDa and 13 kDa peptides. Even the small tryptic fragments reacted with antiphosphatase antibodies from rabbit serum and with monoclonal antibody mAb-14. Papain treatment under these conditions resulted in the release of a 40 kDa peptide which was gradually reduced to a 18 kDa peptide. The monoclonal antibody mAb-14 to the prostatic phosphatase was bound exclusively to the 50 kDa subunit of the phosphatase and to the 40 kDa peptide. The results suggest that the monoclonal antibody mAb-14 binding site represents a "local" sequence rather than a "conformational" one and does not require an extensive tertiary folding of the antigen molecule.     

Development of rules for vaccine engineering based on the variability of peptide fragments in closely related proteins

Based on the protein sequence data bank (PIR), the "variable fragment" bank, comprising pairs of closely related proteins, containing one or more strongly differing sites of primary structures was formed. The bank includes 465 "variable fragments" of 383 protein pairs. Amino acid residues composition of "variable fragments" was examined and indexes of potential amino acid residues variability was formed. An analysis of amino acid fragments replaceability was carried out by substituting the N-, C-terminal, or middle part of a chain), the fragments length differences and physico-chemical properties of residues, such as volume, hydrophobicity, polarity, isoelectric point, etc. Some general empirical rules of peptide insertions in carrier-proteins were created based on these analyses. The rules are directed for performing modifications maintaining the common structure and function of the carrier-protein molecule. The selection scheme for determining the regions suitable for modification and the criteria for defining the width of acceptable modifications in this regions were suggested. The use of potential variability profile for detecting regions suitable for peptide insertion was considered on the model of hepatitis B surface protein.     

Structurally functional organization of corticotropin: lipolytic and steroidogenic activity of some of its fragments]

The influence of ACTH fragments, possessing structural elements, common for certain groups of peptide hormones and kinins--"common" fragments and cluster of basic amino-acids--(Lys 17,18-ACTH 11-18-NH2--I; ACTH 11-13-NH2--II; NH2CO-ACTH18-20-NH2--III) on lipolytic effect of ACTH in rat isolated fat cells and on the steroidogenic effect of ACTH in isolated rat adrenal cells was studied. Fragment I exerts a steroidogenic effect (alpha=0,84) at concentrations of 1--100 microng/ml. At low concentrations (10(-8)--10(-3) microng/ml) fragment I potentiates ACTH-induced steroidogenesis. Fragment I has no effect on the lipolysis;however, it potentiates ACTH-induced lipolysis at concentrations of 10--100 microng/ml. The results obtained support our previous supposition that "common" fragments are essential secondary non-specific active sites of hormones.     

Presence of a prepiece at the NH2-terminal end of pre-cytochrome P-450(SCC) peptide

Mild acid treatment of in vitro translated cytochrome P-450(SCC) (pre-P-450(SCC] peptide cleaved the peptide into two fragments. Comparison of the sizes and the NH2-terminal amino acids of the fragments with those of the corresponding fragments from mature P-450(SCC) suggested that the prepiece of pre-P-450(SCC) was present at the NH2-terminal end of the peptide. This conclusion was confirmed by radio-sequencing of the NH2-terminal portion of pre-P-450(SCC).     

Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the fibronectins

Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the alpha- and beta-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second "cell-binding" domain in the sequences represented by f110 that is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the alpha-subunit and f155 from the beta-subunit--both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44 + 47 having the "extra domain" characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44 + 47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of histones H1o,H5 and HMG proteins on cell division of cultured murine erythroleukemia cells

Summary In the present study the effect of histones H1^o and H5, and the nonhistone chromatin proteins HMG 1, 2, 14 and 17 (the high mobility group proteins), as well as the acidic peptide fragments of HMG 1 and 2 and polyglutamate, on cell division and differentation of cultured murine erythroleukemia (Friend) cells has been investigated. It was found that histones H1^o and H5, the acidic peptide fragments of HMG 1 and 2, HMG 14 and 17 and sodium polyglutamate stimulated cell division at a concentration of 10 g/ml. None of the H1^o, H5 or HMG protein preparations induced hemoglobin synthesis, as judged by benzidine staining.     

Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides

Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.     

Epitopes on proliferating cell nuclear antigen recognized by human lupus autoantibody and murine monoclonal antibody

The immune epitopes of proliferating cell nuclear antigen (PCNA), also called cyclin, were analyzed by determining the reactivity between PCNA peptide fragments and anti-PCNA antibodies from lupus patients, murine monoclonal antibody (19A2), and rabbit anti-NH2-terminal peptide antibody. Limited digestion of PCNA/cyclin with Staphylococcus aureus V8 protease resulted in several peptide fragments. Five fragments of 30, 20, 15, 14, and 13 kDa were reactive with rabbit anti-NH2-terminal peptide antibody denoting that they contained the NH2-terminal peptide. The 30- and 20-kDa fragments reacted with 19A2 but the others did not. Lupus sera reacted with 17- and 15-kDa peptide fragments allowing their classification into three groups. Two of eight sera (type A) reacted only with the 17-kDa fragment. Two others (type B) reacted with both the 17- and 15-kDa fragments and the remaining four sera (type C) reacted only with the 15-kDa fragment. The sera reacting with the 15-kDa fragment also reacted with the 20-kDa fragment, but the sera reactive only with the 17-kDa fragment did not, indicating that the 17-kDa fragment was not a degradation product of 20-kDa fragments. The 19A2 epitope resided in the region between 15 and 20 kDa from the NH2 terminus, whereas there was at least one distinct epitope on each 15- and 17-kDa peptide, which were recognized by lupus autoantibodies.     

Quantification of phosphoproteins with global internal standard technology

Global internal standard technology (GIST) is being developed for the quantification of all primary structure and post-translational variants of proteins in a proteome. This paper is directed at an analysis of phosphorylation, primarily of serine and threonine. Quantification was achieved by acylation of primary amino groups in peptide cleavage fragments of proteins with isotopically coded derivatizing agents. Peptides from controls were globally coded with an isotopically "light" form of the reagent while those from experimental samples were coded with a "heavy" form of the reagent. The two types coding reagents used in this work were N-hydroxyl succinimide derivatives of acetate and 4-trimethylammoniumbutyrate. Heavy isotope forms were produced by deuteration of methyl groups. Subsequent to coding and mixing, the two samples were passed through a Ga(III) immobilized metal affinity chromatography (IMAC) column and the selected peptide fraction was further resolved by reversed-phase chromatography (RPC) and analyzed by mass spectrometry (MS). Relative differences in phosphopeptide concentration between samples were derived from isotope ratio measurements of the peptide isoforms observed in mass spectra. The method was validated with model peptides.     

Cu(II) interaction with N-terminal fragments of human and mouse beta-amyloid peptide

The stoichiometry, stability constants and solution structure of the complexes formed in the reaction of copper(II) with N-terminal fragments of human and mouse beta-amyloid peptide, 1-6, 1-9, 1-10 have been determined by potentiometric, UV/VIS, CD and EPR spectroscopic methods. The fragments 1-9 and 1-10 form complexes with the same coordination modes as the fragments 1-6. The coordination of the metal ion for human and mouse fragments starts from the N-terminal Asp residue which stabilizes significantly the 1N complex as a result of chelation through the beta-carboxylate group. In a wide pH range of 4-10, the imidazole nitrogen of His(6) is coordinated to form a macrochelate. Results show that, in the pH range 5-9 the human fragments form the complex with different coordination mode compared to that of the mouse fragments. The low pK(1)(amide) values (approximately 5) obtained for the mouse fragments may suggest the coordination of the amide nitrogen of His(6) while in case of the human fragments the coordination of the amide nitrogen of Ala(2) is suggested. The replacement of glycine by the arginine residue in the fifth position of the beta-amyloid peptide sequence changes the coordination modes of a peptide to metal ion in the physiological pH range. In a wide pH (including physiological) range the mouse fragments of beta-amyloid peptide are much more effective in Cu(II) binding than the human fragments.     

Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein

The conformational conversion of the nonpathogenic "cellular" prion isoform into a pathogenic "scrapie" protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a beta-like structure. A peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a beta-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152-156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.