Formation and characterization of a single Trp-Trp cross-link in indolicidin that confers protease stability without altering antimicrobial activity.

Indolicidin is a 13-residue cationic, antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils. The unique composition of indolicidin distinguishes it from alpha-helical and beta-structured cationic peptides, because five of indolicidin's 13 residues are tryptophans: H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH(2). Solid phase synthesis of indolicidin gave rise to a minor byproduct that possessed unusual fluorescence and UV absorbance properties compared with authentic indolicidin. The byproduct was purified by combined ion exchange and reversed phase high pressure liquid chromatography steps and was shown be identical to authentic indolicidin in its microbicidal activity against Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. Mass analysis of the byproduct revealed a 2-atomic mass unit reduction compared with indolicidin, suggesting the deprotonation of two indole side chains to form an intrachain delta(1),delta(1)'-ditryptophan derivative. We confirmed the nature of the cross-linked byproduct, termed X-indolicidin, by absorbance and fluorescence spectroscopy, peptide mapping, and sequence analysis. Edman degradation revealed that Trp-6 and Trp-9 were covalently cross-linked. Compared with indolicidin, X-indolicidin was partially resistant to digestion with trypsin and chymotrypsin, suggesting that the ditryptophan stabilizes a subset of molecular conformations that are protease resistant but that are absent in the native structure.

Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Abeta1-42.

Our early study indicates that intracellular Abeta1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Abeta1-42 also leads to the accumulation of newly synthesized intracellular Abeta. Here we describe that newly synthesized Abeta, especially Abetan-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Abeta1-42 treatment. These results suggest that intracellular Abeta may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Abeta1-40, the solid-phase intracellular pathway preferentially produces Abetan-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Abeta also share the same types of Abeta structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.

On the affinity between the plasminogen activator inhibitor type 2 and apolipoprotein A1.

The plasminogen activator inhibitor type 2 (PAI-2) is present in its high molecular weight, glycosylated form in pregnancy plasma. When the protein was purified from retroplacental blood by immunoaffinity chromatography on a PAI-2 antibody column and the retained material was further fractionated by gel filtration chromatography, it was always contaminated by apolipoprotein A1, the latter protein being identified by its N-terminal sequence, molecular weight in SDS-PAGE and immunological properties. The co-purification of the two proteins seemed to indicate a strong affinity between them, suggesting apolipoprotein A1 to be a carrier protein for this PAI-2 form. Further investigation to check this hypothesis showed that the binding of apolipoprotein A1 to the immunoaffinity support was PAI-2-independent and caused by a general surface affinity. This finding was corroborated by a study of the microtitre plate binding properties of the proteins. Pure, high molecular weight PAI 2 did not bind to apolipoprotein A1-coated wells, but the latter protein bound to coated as well as to uncoated wells. Thus, there is no evidence for a specific binding between the two proteins.

Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen.

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.

Characterization of a Borna disease virus glycoprotein, gp18.

Borna disease virus is a nonsegmented negative-strand RNA virus that causes neurologic disease in a wide variety of animal hosts. Here we describe identification and characterization of the first glycoprotein in this viral system. The 18-kDa glycoprotein, gp18, has been purified from infected rat brain. Isolation and microsequencing of this protein allowed identification of a 16.2-kDa open reading frame in the viral antigenome. Lectin binding and endoglycosidase sensitivity assays indicate that gp18 is an unusual N-linked glycoprotein.

Linkage disequilibrium relationships among four polymorphisms within the human fibrinogen gene cluster.

The extent of linkage equilibrium was estimated among four recently characterized human fibrinogen restriction fragment length polymorphisms (RFLPs) using a randomly selected group of 110 individuals from California. Two coding region RFLPs, RsaI and MnlI (FGA codon 312 and FGB codon 448, respectively), and two RFLPs located in the 5' flanking region of the FGB gene, AluI (HindIII) and HaeIII, were analyzed. Maximum likelihood estimates based on genotypic data indicated that the RsaI polymorphism in the FGA gene was at apparent linkage equilibrium with the MnlI, AluI, and HaeIII sites in the FGB gene, but strong linkage disequilibrium was noted for the MnlI-AluI, MnlI-HaeIII, and AluI-HaeIII RFLP pairs within the latter gene. The discrepancy in disequilibrium relationships among these closely linked RFLPs may indicate a region of increased recombination between the FGA and FGB RFLP loci. The FGA RsaI polymorphism, when used in conjunction with any of the FGB sites examined, will provide more detailed linkage or association data than analyses that would utilize only FGB sites. Effective use of polymorphisms within the fibrinogen locus will aid analysis of the relationships between fibrinogen genotype, plasma fibrinogen levels, and risk of cardiovascular disease.

Role of the alpha C domains of fibrin in clot formation.

The role of the carboxyl-terminal portion of the alpha chains of fibrin (alpha C domains) in clot formation was investigated by transmission and scanning electron microscopy and turbidity studies of clots made from preparations of molecules missing one or both of these domains. Highly purified and entirely clottable preparations of bovine fragment X monomer, one containing primarily molecules missing a single alpha C domain (fragment X1) and the other consisting of molecules missing both alpha C domains (fragment X2), were used for these experiments. These preparations were characterized by various methods, including the complete determination of the amino- and carboxyl-termini of all peptides and fragments. These preparations formed clots on dilution to neutral pH. In all cases, clots observed by either scanning or transmission electron microscopy were made up of a branched network of fibers, similar to those formed by thrombin treatment of intact fibrinogen, suggesting that the alpha C domains are not necessary for protofibril and fiber formation or branching. However, both the fiber and clot structure varied with the different fractions, indicating that the alpha C domains do participate in polymerization. The rate of assembly, as indicated by the lag period and maximum rate of turbidity increase, as well as the final turbidity, was decreased with removal of the alpha C domains, suggesting that they accelerate polymerization. preparations of isolated alpha C fragment added to fibrin monomer have striking effects on the turbidity curves, showing a decrease in the rate of polymerization in a dose-dependent manner but not complete inhibition. Electron microscopy of fibrin monomer desA molecules at neutral pH showed that most of the alpha C domains, like those in fibrinogen, remain associated with the central region. Thus, it appears that normally with thrombin cleavage of fibrinogen the effects of the interactions of alpha C domains observed here will be most significant for lateral aggregation.

A proposal for a coherent mammalian histone H1 nomenclature correlated with amino acid sequences.

Bio-Rex 70 chromatography was combined with reverse-phase (RP) HPLC to fractionate histone H1 zero and 4 histone H1 subtypes from human placental nuclei as previously described (Parseghian MH et al., 1993, Chromosome Res 1:127-139). After proteolytic digestion of the subtypes with Staphylococcus aureus V8 protease, peptides were fractionated by RP-HPLC and partially sequenced by Edman degradation in order to correlate them with human spleen subtypes (Ohe Y, Hayashi H, Iwai K, 1986, J Biochem (Tokyo) 100:359-368; 1989, J Biochem (Tokyo) 106:844-857). Based on comparisons with the sequence data available from other mammalian species, subtypes were grouped. These groupings were used to construct a coherent nomenclature for mammalian somatic H1s. Homologous subtypes possess characteristic patterns of growth-related and cAMP-dependent phosphorylation sites. The groupings defined by amino acid sequence also were used to correlate the elution profiles and electrophoretic mobilities of subtypes derived from different species. Previous attempts at establishing an H1 nomenclature by chromatographic or electrophoretic fractionations has resulted in several misidentifications. We present here, for the first time, a nomenclature for somatic H1s based on amino acid sequences that are analogous to those for H1 zero and H1t. The groupings defined should be useful in correlating the many observations regarding H1 subtypes in the literature.

Human fetal spinal cord xenografted to the eye of athymic nude rats: survival, ultrastructural differentiation, glial responses and vascular interactions.

The ultrastructure of human spinal cord xenografts transplanted to the anterior chamber of athymic nude rats was studied. The grafts displayed long-term survival and expressed several organotypical characteristics of normal human spinal cord. Three cell types of different sizes--small, intermediate, and large--and two different types of neuropil, with and without myelinated axons, were observed in the central cellular layer. The myelin-free area was formed between and round small neurons and was composed of many nonmyelinated fibers and bundles of fine axons. The hostgraft interface, especially around vessels from host iris, exhibited marked interdigitations of astroglial processes and basement membranes. Thick, superficial glial layers containing many fibrous astrocytes and many glial processes were also seen in both the intermediate axonal and central cellular layers. Thin-walled vessels were clustered in the central layer, and their perivascular spaces contained collagen fibers. No central nervous system-type vessels were observed. In conclusion, both the organotypical features of normal spinal cord and the glial responses and abnormal vascular interactions observed in this study must be considered in relation to the long-term survival and functional potential of xenografted neural tissue.

Covalent structure of botulinum neurotoxin type A: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains.

Botulinum neurotoxin Type A is synthesized by Clostridium botulinum as a approximately 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a approximately 50-kD light chain and a approximately 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequence predicted a total of 9 Cys residues (Binz et al., 1990, J. Biol. Chem. 265, 9153-9158; Thompson et al., 1990, Eur. J. Biochem. 189, 73-81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residues predicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridge connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Human fibrinogen polymorphic site analysis by restriction endonuclease digestion and allele-specific polymerase chain reaction amplification: identification of polymorphisms at positions A alpha 312 and B beta 448.

In the fibrinogen molecule, a total of seven sites have been tentatively identified as polymorphic; however, disagreements about these sites have been observed among the various protein and DNA sequence data published. To allow examination of the potential polymorphic sites at the DNA level, human genomic DNA samples were prepared from 110 unrelated, healthy individuals. Either allele-specific polymerase chain reaction (ASPCR) amplification or PCR amplification followed by restriction endonuclease digestion was used to detect the presence of possible polymorphisms. Two polymorphic sites were confirmed, one at A alpha 312 (Thr/Ala) by RsaI restriction analysis, and a second at B beta 448 (Arg/Lys) by MnlI restriction analysis. Mendelian inheritance of both polymorphisms was demonstrated and allele frequencies were estimated as 0.76/0.24 and 0.85/0.15 for the A alpha 312 and B beta 448 sites, respectively. The sites at A alpha 47, A alpha 296, B beta 162, B beta 296, and gamma 88 showed no evidence of variation in any of our samples. The amino acid polymorphisms at A alpha 312 and B beta 448 reflect conservative residue changes with unknown effects on fibrinogen structure or function. An additional, previously unrecognized DNA sequence variant was detected in a single individual in the second intron of the A alpha chain using HinfI restriction analysis.

Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils.

A new family of cysteine-rich antimicrobial peptides from bovine neutrophils was isolated and characterized. Thirteen structurally homologous peptides were purified to homogeneity from a granule-rich cytoplasmic fraction of purified blood neutrophils. The complete sequences of the peptides were determined by a combination of enzymatic digestion, Edman degradation, and additional biochemical characterization of the carboxyl termini. The peptides are characterized by a highly cationic 38-42-residue chain which includes 6 invariantly spaced cysteines which form three disulfides. They share a highly conserved consensus sequence which is also found in a recently described epithelial antimicrobial peptide from bovine trachea. The in vitro antibacterial activities of the 13 neutrophil peptides, determined in assays using Staphylococcus aureus and Escherichia coli as test organisms, demonstrated that each peptide possessed antimicrobial activity, and that several were as active as the most potent neutrophil defensin, rabbit NP-1. Though the structural and functional attributes of the bovine neutrophil peptides are similar to those of defensins, the two peptide families are distinguished by their unique consensus sequences and additionally by differing tridisulfide motifs. We therefore propose that this new defensin-like antimicrobial peptide family be named beta-defensins.

Nerve growth factor-induced excitation of selected neurons in the brain which is blocked by a low-affinity receptor antibody.

We have investigated the electrophysiological effects of nerve growth factor (NGF) on single-neuron activity in central nervous system (CNS) grafts of septum, spinal cord, and hippocampus in oculo. NGF was found to have slow-onset, long-lasting excitatory effects on the spontaneous firing of neurons in septal grafts, while no such effects were found in neurons of either hippocampal or spinal cord grafts. Pretreatment with an antibody against the p75 low-affinity NGF receptor blocked the NGF-induced excitations. A second NGF application caused much stronger excitatory responses in sensitive neurons. Our data suggest that forebrain cholinergic neurons may be selectively sensitive to NGF also at the neurophysiological level, responding by excitations, and that NGF upregulates these responses within less than an hour.

Evidence for two distinct major protein components, PAR 1 and PAR 2, in the paraflagellar rod of Trypanosoma cruzi. Complete nucleotide sequence of PAR.

The previously identified major protein components of the paraflagellar rod in Trypanosoma cruzi, PAR 1 and PAR 2, were analyzed to determine if they are distinct proteins or different conformations of a single polypeptide as has been suggested for other trypanosomatids. Amino acid sequence analysis showed PAR 1 and PAR 2 to be two distinct polypeptides. Antibodies specific against either PAR 1 or PAR 2 were shown to each react with a distinct band in Western blots of paraflagellar isolates of T. cruzi and other trypanosomatids if rigorous protease inhibition was used. The PAR 2 message was isolated and characterized by Northern blot and nucleic acid sequence analysis. Preliminary analysis of the PAR 2 gene indicates that PAR 2 is a member of a multigene family with all members residing on a single chromosome.

Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase.

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.

Enteric defensins: antibiotic peptide components of intestinal host defense.

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.

The type II isoform of bovine brain protein L-isoaspartyl methyltransferase has an endoplasmic reticulum retention signal (...RDEL) at its C-terminus.

Bovine brain is known to contain two major isoforms of protein L-isoaspartyl methyltransferase (PIMT), an enzyme that facilitates repair of atypical L-isoaspartyl peptide bonds in proteins. Although the two isoforms can be separated by anion-exchange chromatography, they appear to have similar, if not identical, substrate specificities in vitro. The more basic type I isoform has been extensively characterized, and its complete sequence has been reported. The present study was undertaken in an attempt to understand the structural and functional uniqueness of the more acidic type II isoform. Electrospray mass spectrometry of the intact enzymes revealed that the type II isoform is approximately 43 amu heavier than the type I isoform. Cyanogen bromide cleavage followed by HPLC with on-line mass analysis revealed that the type II isoform contains a unique C-terminal fragment which is 43 amu heavier than the corresponding fragment from the type I isoform. Amino acid composition analysis and direct sequencing of this fragment indicate that the type II isoform ends in the sequence ...RDEL, while the type I is known to end in ...RWK. Since ...RDEL, like ...KDEL, serves as an effective endoplasmic reticulum retention signal, we propose that the type II isoform serves to repair damaged proteins within the endoplasmic reticulum or, perhaps, within some other specialized compartment of the cell. Comparison of the protein sequences of the two bovine brain isoforms to DNA sequences for rodent PIMT reported by others suggests that the type II isoform may be produced by splicing within the codon for Arg224.