Fragment Of Alpha Chain Research Articles

Application Value of Mass Spectrometry in the Differentiation of Benign and Malignant Liver Tumors.

BackgroundDifferentiation of malignant from benign liver tumors remains a challenging problem. In recent years, mass spectrometry (MS) technique has emerged as a promising strategy to diagnose a wide range of malignant tumors. The purpose of this study was to establish classification models to distinguish benign and malignant liver tumors and identify the liver cancer-specific peptides by mass spectrometry.Material/MethodsIn our study, serum samples from 43 patients with malignant liver tumors and 52 patients with benign liver tumors were treated with weak cation-exchange chromatography Magnetic Beads (MB-WCX) kits and analyzed by the Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF-MS). Then we established genetic algorithm (GA), supervised neural networks (SNN), and quick classifier (QC) models to distinguish malignant from benign liver tumors. To confirm the clinical applicability of the established models, the blinded validation test was performed in 50 clinical serum samples. Discriminatory peaks associated with malignant liver tumors were subsequently identified by a qTOF Synapt G2-S system.ResultsA total of 27 discriminant peaks (p<0.05) in mass spectra of serum samples were found by ClinPro Tools software. Recognition capabilities of the established models were 100% (GA), 89.38% (SNN), and 80.84% (QC); cross-validation rates were 81.67% (GA), 81.11% (SNN), and 86.11% (QC). The accuracy rates of the blinded validation test were 78% (GA), 84% (SNN), and 84% (QC). From the 27 discriminatory peptide peaks analyzed, 3 peaks of m/z 2860.34, 2881.54, and 3155.67 were identified as a fragment of fibrinogen alpha chain, fibrinogen beta chain, and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), respectively.ConclusionsOur results demonstrated that MS technique can be helpful in differentiation of benign and malignant liver tumors. Fibrinogen and ITIH4 might be used as biomarkers for the diagnosis of malignant liver tumors.

Low-mass-ion discriminant equation (LOME) for ovarian cancer screening

BackgroundA low-mass-ion discriminant equation (LOME) was constructed to investigate whether systematic low-mass-ion (LMI) profiling could be applied to ovarian cancer (OVC) screening.ResultsMatrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed to obtain mass spectral data on metabolites detected as LMIs up to a mass-to-charge ratio (m/z) of 2500 for 1184 serum samples collected from healthy individuals and patients with OVC, other types of cancer, or several types of benign tumor. Principal component analysis-based discriminant analysis and two search algorithms were employed to identify discriminative low-mass ions for distinguishing OVC from non-OVC cases. OVC LOME with 13 discriminative LMIs produced excellent classification results in a validation set (sensitivity, 93.10 %; specificity, 100.0 %). Among 13 LMIs showing differential mass intensities in OVC, 3 metabolic compounds were identified and semi-quantitated. The relative amount of LPC 16:0 was somewhat decreased in OVC, but not significantly so. In contrast, D,L -glutamine and fibrinogen alpha chain fragment were significantly increased in OVC compared to the control group (p = 0.001 and 0.002, respectively).ConclusionThe present study suggested that OVC LOME might be a useful non-invasive tool with high sensitivity and specificity for OVC screening. The LOME approach could enable screening for multiple diseases, including various types of cancer, based on a single blood sample. Furthermore, the serum levels of three metabolic compounds—D,L -glutamine, LPC 16:0 and fibrinogen alpha chain fragment—might facilitate screening for OVC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13040-016-0111-7) contains supplementary material, which is available to authorized users.

Potential Biomarker Peptides Associated with Acute Alcohol-Induced Reduction of Blood Pressure.

The purpose of this study was to explore the peptides that are related to acute reduction of blood pressure after alcohol drinking. Venous blood was collected from male healthy volunteers before and after drinking white wine (3 ml/kg weight) containing 13% of ethanol. Peptidome analysis for serum samples was performed using a new target plate, BLOTCHIP®. Alcohol caused significant decreases in systolic and diastolic blood pressure levels at 45 min. The peptidome analysis showed that the levels of three peptides of m/z 1467, 2380 and 2662 changed significantly after drinking. The m/z 1467 and 2662 peptides were identified to be fragments of fibrinogen alpha chain, and the m/z 2380 peptide was identified to be a fragment of complement C4. The intensities of the m/z 2380 and m/z 1467 peptides before drinking were associated with % decreases in systolic and diastolic blood pressure levels at 45 min after drinking compared with the levels before drinking, while there were no significant correlations between the intensity of the m/z 2662 peptide and % decreases in systolic and diastolic blood pressure levels after drinking. The m/z 1467 and 2380 peptides are suggested to be markers for acute reduction of blood pressure after drinking alcohol.

Alterations in a Panel of Serum Peptides Correlate with the Disease States of Multiple Myeloma

Background: Multiple myeloma (MM) is prone to misdiagnosis and missed diagnosis in early stage due to heterogeneity of clinical manifestations. Previously, we constructed the MM Supervised Neural Network Algorithm (SNN) diagnostic model based on four peptides through proteomic profiling. The current study is to validate the efficacy of the diagnostic model in a larger size of sample, to verify the identity of proteins the four peptides represent, and to characterize the expression patterns of these proteins in different disease states of MM.Methods: Patients hospitalized in the Second Affiliated Hospital of Xi'an Jiaotong University from January 2010 to December 2011 were enrolled for the study, including 62 Newly diagnosed(ND) MM (age range 44-73, median age 56.5 years old, male/female 34/28), 38 complete remission and very good partial remission (CR&VGPR) MM (age range 44-71, median age 54.6 years old, male/female 21/17) and 43 refractory & relapsed (RR) MM (age range 45-73, median age 57.5 years old, male/female 27/16) after two courses of chemotherapy. 64 healthy controls (HC) (age range 45-70, median age 57.5 years old, male/female 34/30) were recruited from those who came to our hospital for physical examination. No abnormal symptoms or test results were found in HC. Weak cation exchange magnetic beads combined with MALDI-TOF-MS were to analyze serum peptide profiling of MM in different disease groups. We utilized a nano-LC/ESI-MS/MS system to perform peptide sequencing and peptide identification. Immunoblotting and ELISA were performed for validation. ANOVA test was used to analyze differences in different groups. Correlation analysis was performed using linear regression model. Statistical significance was defined as p<0.05 with assumption of equally deviation and two-sided distribution. For multiple comparisons among the four groups, significant level was adjusted to 0.0083(0.05/4(4-1)/2).Results: 58/62 ND cases of MM were correctly assigned to MM group (93.55% sensitivity), while 59 HC were correctly identified as the non-diseased (92.19% specificity). The relative intensities of peptides with molecular weight of 2660.65, 2900.4 and 3315.96 Da were increased in the ND and RR groups when comparing with the CR&VGPR group and HC group(p=0.0000505; p<0.000001; p<0.000001), but there was no significant difference between the two groups for any of the peptides (p=0.14; p=0.25; p=0.21). No significant difference was found between the CR & VGPR group and HC group (p=0.52; p=0.24; p=0.15).The relative intensities of peptide with molecular weight of 7763.24 Da were reduced in the ND group and RR group when comparing with the CR&VGPR group and HC group (p<0.000001), and the two groups had no significant difference (p=0.37). No significant difference was observed between the HC group and CR&VGPR group (p=0.46). The four peptides were identified as fragments of fibrinogen alpha chain (FGA), dihydropyrimidinase- like 2, alpha-fetoprotein and PF4, respectively. Western blot results showed that levels of the FGA and dihydropyrimidinase-like 2 differ between the MM and normal cells (p=0.0034, p=0.0067). No or weak band representing PF4 was seen in ND and RR MM cases. Quantification of bands from western blot analysis showed a significantly decreased level of PF4 in MM-ND and RR samples, relative to age-matched MM-CR&VGPR and HC samples (p=0.0031). The contents of PF4 in 62 ND MM (0.67± 0.18μg/L) was significantly lower compared to 64 HC (1.43 ± 0.77μg/L) (p=1.86 E-6).The FGA contents of ND MM (276.78 ± 194.75μg/L) and HC (30.44 ± 22.15μg/L) differed significantly (p=1.82E-12). The serum VEGF contents of ND MM (669.69 ± 137.81ng/L) were significantly higher compared to HC (120.75 ± 36.96ng/L) (p=7.28E-29). No significant difference of PF4 content was found between thrombocytopenia group (0.65 ± 0.18μg/L) and platelet normal group (0.69 ± 0.17μg/L) in MM (p=0.55). Linear regression analysis showed no correlation between PF4 content and platelet count (correlation coefficient=0.18, p=0.17), whereas serum PF4 and serum VEGF were negatively correlated (correlation coefficient=-0.96, p=1.11E-14).Conclusion: The MM SNN model has provided encouraging efficiency in discriminating MM from HCs. The close correlation of the four peptides with MM disease states pointed to their potential value in monitoring MM treatment response. DisclosuresNo relevant conflicts of interest to declare.

AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity

Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the thimet oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides. Indeed, we have identified the peptide AGHLDDLPGALSAL (AGH), a fragment of the hemoglobin alpha-chain, which specifically bind to the inactive thimet oligopeptidase in the substrate capture assay. Previous peptidomics studies have identified the AGH as well as many other natural peptides derived from hemoglobin alpha-chain containing this sequence, further suggesting that AGH is a natural endogenous peptide. Pharmacological assays suggest that AGH inhibits peripheral inflammatory hyperalgesic responses through indirect activation of mu opioid receptors, without having a central nervous system activity. Therefore, we have successfully used the substrate capture assay to identify a new endogenous bioactive peptide derived from hemoglobin alpha-chain.

Isolation and characterization of a novel antibacterial peptide derived from hemoglobin alpha in the liver of Japanese eel, Anguilla japonica

We isolated and characterized a novel antibacterial peptide, AJHbα, derived from hemoglobin alpha in the liver of Japanese eel, Anguilla japonica. It with concentration of 11.30 μM exhibited stronger antibacterial activity against pathogenic bacterium 1 × 106 cell ml−1Edwardsiella tarda than other two bacteria. The extraction procedure for AJHbα included extraction with acetate acid, ultrafiltration, cation-exchange chromatography on HiTrap™ CM FF, reverse-phase liquid chromatography on Source 5R RPC and C18 RP-HPLC. MALDI-TOF MS suggested that the peptide had an observed molecular weight of 2388.05 Da. Its amino acid sequence determined by Edman degradation was similar to those of hemoglobin alpha chain in other fish by BLAST analysis. A complete N-terminal amino acid sequence of the AJHbα was FAHWPDLGPGSPSVKKHGKVIM corresponding to the cDNA sequence by RACE amplification. Its synthetic peptide had strong antibacterial activities against ten Gram-positive or negative bacteria. To our knowledge, AJHbα was the first identified fragment of hemoglobin alpha chain with strong antibacterial activity in fish.

A new sandwich biomarker assay for cartilage type ii collagen can discriminate between no osteoarthritis (oa), pre-radiographic and radiographic knee oa in human urine

A new sandwich biomarker assay for cartilage type ii collagen can discriminate between no osteoarthritis (oa), pre-radiographic and radiographic knee oa in human urine

Serum Degradome Markers for the Detection of Breast Cancer

Many proteins have been proposed as potential biomarkers for breast cancer. Yet, validation of their discriminative value using quantitative methods has scarcely been performed. In this study, we investigated the discriminative value of six peptides that were previously proposed to be generated by breast cancer specific exoproteases: bradykinin, des-Arg(9)-bradykinin, Hyp(3)-bradykinin, and fragments of fibrinogen alpha-chain (Fib-alpha ([605-629])), complement component 4a (C4a ([1337-1350])), and interalpha trypsin inhibitor heavy chain 4 (ITIH4 ([666-687])). Their absolute serum concentrations were measured with a completely validated liquid chromatography-tandem mass spectrometric assay (LC-MS/MS) and compared between 62 newly diagnosed breast cancer patients and 62 controls matched for age and sample storage duration. Both ITIH4 ([666-687]) and des-Arg(9)-bradykinin showed statistically significantly higher median concentrations in breast cancer samples than in matched control samples. Additionally, we analyzed serum samples collected after surgical removal of the tumor, in which median ITIH4 ([666-687]) and des-Arg(9)-bradykinin concentrations were significantly decreased and not statistically significantly different from concentrations in the controls anymore. In a combined analysis, ITIH4 (666-687]) and des-Arg(9)-bradykinin independently contributed to the discrimination between cases and controls. In this study, we confirmed that the exoprotease breakdown peptides, ITIH4 (666-687]) and des-Arg(9)-bradykinin, differed between breast cancer cases and controls, supporting the potential of degradome markers for the diagnosis of breast cancer.

Comprehensive analysis of short peptides in sera from patients with IgA nephropathy

We analyzed serum short peptides comprehensively to know whether they were useful to characterize IgA nephropathy (IgAN). Serum samples from 26 patients with untreated IgAN and 25 healthy donors were tested. Short peptides with molecular weights of approximately 7 kDa, purified from the serum samples by magnetic-beads-based weak cation exchange, were detected by mass spectrometry. Then the peptide peaks detected were subjected to the multivariate data analysis by SIMCA-P+ containing principal component analysis (PCA) and orthogonal partial-least-squares-discriminate analysis (OPLS-DA). A total of 92 peptide peaks were detected in the tested serum samples. The OPLS-DA analysis revealed that the profile of all the peptide peak intensities discriminated the IgAN group and the healthy group completely with a high R2 value (0.919) and a high Q2 value (0.861). Further, the profile of only five peptide peaks was found to discriminate the two groups. By tandem mass spectrometry and database searching, three of the five peptides which increased in the IgAN group were identified as fragments of fibrinogen alpha chain, and the two peptides which increased in the healthy group were identified as fragments of complement C3f and kininogen-1 light chain. Taken together, the profile of the serum short peptides would be useful to discriminate IgAN and healthy conditions. Further, the five peptides may be candidate serum markers for IgAN and may be related to pathogenesis of IgA.

Stimulation of fibroblast proliferation by neokyotorphin requires Ca2+ influx and activation of PKA, CaMK II and MAPK/ERK

Neokyotorphin [TSKYR, hemoglobin alpha-chain fragment (137-141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4beta-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+ L-type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin.

Derivatives of N-amidinoproline and their use in conventional and solid phase peptide synthesis

N-Amidinoproline, a hybrid structure modeling key features of the Arg-Pro sequence, was synthesized. The activation of carboxyl group of free N-amidinoproline was found to result in the formation of a cyclic side product, whose structure was confirmed by ESI MS, 1H NMR, and 13C NMR spectra. The preparation of N-(mesitylenesulfonylamidino)-L-proline using the mesitylenesulfonyl derivative of 2-methylisourea was demonstrated to be accompanied by partial racemization. The target product was synthesized by modification of N-amidinoproline by mesitylenesulfonyl chloride. The possibility of using N-amidinoproline in the N-terminal modification of a peptide chain was shown by the example of synthesis of an analogue of the 95-98 fragment of fibrinogen alpha chain.

Association of citrullinated proteins with synovial exosomes

In addition to releasing proteins and mediators, cells also release membrane vesicles (exosomes and apoptotic blebs) into the extracellular environment. Apoptotic blebs contain multiple autoantigens, but few data are available concerning the protein content of exosomes. Exosomes are formed during an immune response and can directly stimulate T cells or bind to dendritic cells. The aim of this study was to identify the nature of synovial exosomes from patients with different rheumatic diseases and to examine their potential autoantigenic content, which may be involved in the induction of an autoimmune response. Synovial exosomes from patients with rheumatoid arthritis (RA), patients with reactive arthritis, and patients with osteoarthritis were purified, analyzed by electron microscopy, and labeled with immunogold to detect IgG and IgM molecules. Autoantigen content was identified by 2-dimensional electrophoresis-immunoblotting and subsequent mass spectrometry. In order to investigate the presence of citrullinated proteins, immunoblotting with anticitrulline antibodies was performed. Citrullinated proteins were observed in all exosome preparations, in contrast to other autoantigenic proteins (e.g., BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases. These citrullinated proteins included the fibrin alpha-chain fragment, fibrin beta-chain, fibrinogen beta-chain precursor, fibrinogen D fragment, and the Sp alpha (CD5 antigen-like protein) receptor. Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Sp alpha associated with IgM and IgG. Synovial exosomes contain citrullinated proteins, which are known to be autoantigens in RA. Although immune mechanisms in which exosomes carry citrullinated peptides could play an important role in the induction and distribution of citrullinated proteins, there must be a specific recognition of these proteins that is unique to the RA immune system.

Novel Natural Peptide Substrates for Endopeptidase 24.15, Neurolysin, and Angiotensin-converting Enzyme

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.

Properties of normal and mutant polypeptide fragments from the dimer self-association sites of human red cell spectrin.

We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the alpha-chain and the C-terminus of the beta-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of two helices from the C-terminal part of the beta-chain and one helix from the N-terminus of the alpha-chain. Three mutant beta-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The alpha- and beta-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold. Unexpectedly, the conformational stability of the folded beta-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible explanations for this phenomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wildtype alpha- and beta-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied by an increase in alpha-helicity. From continuous-variation profiles an association constant in the range 1-2 x 10(6) M-1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus of the spectrin beta-chain. Of the three mutations in the beta-chain fragment, one (an Ala-->Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the alpha-chain fragment, whereas Trp-->Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic conditions associated with the mutations.

Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases.

A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.

Hydrodynamic studies of a complex between the Fc fragment of human IgE and a soluble fragment of the Fc epsilon RI alpha chain.

The interaction between immunoglobulin E (IgE) and its high-affinity receptor Fc epsilon RI is central to allergic disease. The binding site for Fc epsilon RI lies in the third constant region domain of the epsilon heavy chain of IgE (C epsilon 3). Identical epitopes on the two C epsilon 3 domains in the IgE-Fc are predicted to be on opposite sides of the structure, and therefore each could bind independently to a receptor molecule. Titrations, however, reveal that the IgE-Fc forms an equimolar complex with a soluble fragment of the Fc epsilon RI alpha chain (sFc epsilon RI alpha), and the molecular weight of the complex, as determined by sedimentation equilibrium, confirms this stoichiometry. The measured sedimentation coefficients of the two ligands are in good agreement with computed values for a compact IgE-Fc and an elongated sFc epsilon RI alpha structure. The calculated sedimentation coefficients for possible models of a 1:1 complex lead to exclusion of all highly extended geometries for the complex. Possible explanations for the paradoxical stoichiometry of the IgE-Fc/sFc epsilon RI alpha complex, in terms of the curved shape of IgE, a conformational change in IgE when the receptor binds, and steric interference between two molecules of Fc epsilon RI binding to identical sites, are discussed.

Activation of the Alternative Pathway of Human Complement by Apoptotic Human Umbilical Vein Endothelial Cells1

Complement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the alpha chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major alpha chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the alpha 1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its alpha' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.

Allele specificity of structural requirement for peptides bound to HLA-DRB1*0405 and -DRB1*0406 complexes: implication for the HLA-associated susceptibility to methimazole-induced insulin autoimmune syndrome.

Self-peptides bound to HLA-DR4 (DRA-DRB1*0405 complex) were eluted from the purified DR4 complex, fractionated on reverse-phase HPLC, and subjected to NH2-terminal sequencing. Seven independent sequences were obtained, and all putative peptides synthesized bound to DRB1*0405 as well as DRB1*0406 complex, which differ only at DR beta residues 37, 57, 74, and 86. Binding assay using analogue peptides of a DR4 binder GSTVFDNLPNPE revealed that FxxLxN is an important anchor motif necessary for binding (where x is any amino acid), which was common to DRB1*0405 and 0406. Determination of the binding affinity of 60 synthetic AAFAALANAA-based analogue peptides showed that substituting F to W or C; L to F, W, or Y; and N to Q or S on AAFAALANAA changed the affinity substantially between DRB1*0405 and DRB1*0406. It is noteworthy that all patients with methimazole-induced insulin autoimmune syndrome are positive for DRB1*0406 and negative for DRB1*0405. Interestingly, the quantitative structural motif identified in this study predicted that 8TSICSLYQLE17 of human insulin alpha chain may bind specifically to DRB1*0406 using its 10IxxLxQ15 motif. Indeed, DRB1*0406 complex bound 8TSICSLYQLE17 with a high affinity, and in striking contrast, DRB1*0405 complex did not. Furthermore, a short-term T cell line specific to human insulin established from a DRB1*0406-bearing individual did show reactivity with a peptide fragment containing the 10IxxLxQ15 motif. Although this fragment probably exists at a very low level under normal physiological conditions due to the disulfide bond between flanking cysteine residues (6Cys-11Cys), a reducing compound such as methimazole may cleave the disulfide bond in vivo and allow DR alpha-DRB1*0406 complex on antigen-presenting cells to bind much of the linear fragment of insulin alpha chain, which may lead to the activation of self-insulin-specific T-helper cells.

Further studies on the interaction of migrating keratinocytes with fibrinogen.

If glass implants placed under one edge of a skin wound in the adult newt are coated with fibrinogen (FGN), keratinocytes from the wound periphery migrate onto the implant. To learn more about the site(s) in FGN that permits this migration, we exposed keratinocytes to implants coated with forms of FGN containing modifications or deletions in the 3 most commonly studied cell binding sites; the RGDF sequence at A alpha 95-98, RGDS at A alpha 572-575 and the carboxy terminal 12 amino acids in the gamma A chain. Recombinant FGN with either RGD sequence altered to RGE supported migration as well as unmodified FGN did. Replacement of the carboxy terminal 4 amino acids in the gamma A chain by a 20 amino acid sequence that disrupts the ability of the gamma terminus to mediate platelet aggregation (the gamma' variant) likewise had no effect. Nor did simultaneous antibody blockade of the RGDS, RGDF, and gamma A sites have any effect. At its best, Dhem1, a fragment containing the RGDS and gamma A sites, produced only about half as much migration as the maximum obtained on intact FGN. Dhem2, a fragment differing from Dhem1 only by having a gamma' variant in place of gamma A, was even less active. Two other D fragments, both of which were missing a large part of the A alpha chain, and one of which contained none of the three major binding sites, supported considerable migration, suggesting that loss of the A alpha chain COOH terminus reveals a site that was not exposed in Dhem1 and 2. A alpha chain fragments containing the RGDF or RGDS sequence were active, but a much larger fragment without RGD was inactive. A soluble peptide consisting of the sequence, RGDS, was a potent inhibitor of migration on FGN but RGDF and the gamma A pentapeptide, KQAGD, were minimally effective. Longer versions of these peptides decreased the effectiveness in all cases. These results suggest that under certain circumstances, newt keratinocytes may interact with each of the 3 major binding sites in FGN as well as a site outside these sequences.

Amino acid residues 1101-1105 of the isotypic region of human C4B is important to the covalent binding activity of complement component C4.

The C4A and C4B isotypes of human C4 show certain functional differences that stem from their relative preference for transacylation to amino (-NH2) vs hydroxyl (-OH) nucleophiles, respectively, on complement-activating surfaces. Comparison of amino acid sequences of the alpha-chain fragment of C4, C4d, has shown C4A- and C4B-specific sequences at residues 1101-1106 are the only consistent structural difference between isotype, i.e., Pro, Cys, Pro, Val, Leu, Asp in C4A and Leu, Ser, Pro, Val Ile, His in C4B. These residues may be responsible either in part or entirely for properties associated with isotype. To examine the functional role of residues 1101-1106 in C4B-mediated hemolysis, whole serum or immunopurified human C4 with allotypes, A3B1, A3, B2B1, or B1 were preincubated in the presence or absence of an antipeptide mAb (BII-1) specific for amino acid residues 1101-1105 of C4B. Sensitized sheep E and C4-deficient guinea pig serum was then added and lysis measured by absorbance at 415 nm. Our results show lysis of antibody-sensitized sheep E is inhibited by antibody and C4B2B1, C4B1, or C4A3B1 but not antibody and C4A3. The interference of hemolysis by BII-1 could not be explained by inhibition of activation of C4B or inhibition of C3 or C5 convertase activity. Furthermore, results from uptake experiments show that BII-1 interferes with the covalent binding activity of C4B, indicating residues 1101-1105 play a role in the covalent binding reaction of C4B to the target E-antibody complex.