scholarly journals C1 fixation and classical complement pathway activation by a fragment of the Cmu4 domain of IgM.

1975 ◽  
Vol 142 (5) ◽  
pp. 1322-1326 ◽  
Author(s):  
M M Hurst ◽  
J E Volanakis ◽  
R M Stroud ◽  
J C Bennett
Keyword(s):  
Complement Fixation ◽  
Marked Decrease ◽  
Classical Complement Pathway ◽  
Amino Terminal ◽  
Pathway Activation ◽  
Normal Human ◽  
Carboxy Terminal Region ◽  
The Individual ◽  
Carboxy Terminal ◽  
Classical Complement

A 56 residue fragment derived from a Waldenströme IgM protein and consisting of 24 residues of the amino-terminal portion of the Cmu4 domain disulfide bonded to 32 residues of the carboxy-terminal region of the loop has been shown to fix active C1 (C1) in a C1-fixation assay. Cleavage of the disulfide bond within the CH4 fragment resulted in a marked decrease of C1-fixing ability, although the isolated A and B fragments did retain a limited ability to fix C1. Upon incubation with normal human serum the intact CH4 fragment and equal molar amounts of the isolated A and B peptides consumed C4 suggesting that the C1-activating determinant of IgM remains intact in these three fragments. Furthermore, on a molar basis the intact or the reduced CH4 fragment consumed C4 as effectively as each of its component chains suggesting that transient binding of C1 by the individual A and B peptide chains is sufficient to activate C1. On the basis of these observations it is proposed that a classical complement fixation function, i.e. C1 binding and activation, can be localized within a region of the IgM molecule corresponding to the Cmu4 domain.

scholarly journals In vitro complement binding on cytoplasmic structures in normal human skin: immunoelectronmicroscopic studies.

1982 ◽  
Vol 95 (2) ◽  
pp. 543-551 ◽  
Author(s):  
G Schuler ◽  
H Hintner ◽  
K Wolff ◽  
P Fritsch ◽  
G Stingl
Keyword(s):  
Cell Types ◽  
Exact Nature ◽  
Complement Pathway ◽  
Experimental Conditions ◽  
Complement Components ◽  
Classical Complement Pathway ◽  
Pathway Activation ◽  
Normal Human ◽  
Cytoplasmic Structures ◽  
Classical Complement

We have previously provided evidence that suggests that exposure of cryostat skin sections to normal human serum (NHS) results in the antibody-independent Clq binding to cytoplasmic structures of various cell types, leading to classical complement pathway activation as evidenced by cytoplasmic C3 deposition. In the present study, we have employed immunoelectronmicroscopic methods to clarify the exact nature of cytoplasmic C3 binding structures. Incubation of cryostat skin sections with NHS followed by peroxidase-labeled rabbit anti-human C3 serum (HRP-R/Hu C3) revealed that intracytoplasmic binding of C3 occurred in suprabasal keratinocytes, melanocytes, fibroblasts, smooth muscle cells, endothelial cells, pericytes, Schwann cells, and nerve axons, but not in basal keratinocytes, Langerhans cells, and other cellular constituents of the skin. C3 binding, as revealed by the deposition of HRP reaction product, was exclusively confined to intermediate-sized filaments (ISF), which can therefore be considered to represent the subcellular site for classical complement pathway activation. Under experimental conditions that do not allow classical complement pathway activation, ISF were not decorated. Our observation that ISF of ontogenetically different cell types share the capacity of complement fixation is in accordance with the recent finding that different ISF types, despite their biochemical and antigenic heterogeneity, have common alpha-helical domains and may provide a clue to the mechanism and site of interaction between complement components and ISF.

scholarly journals Heptose I Glycan Substitutions on Neisseria gonorrhoeae Lipooligosaccharide Influence C4b-Binding Protein Binding and Serum Resistance

2007 ◽  
Vol 75 (8) ◽  
pp. 4071-4081 ◽  
Author(s):  
Sanjay Ram ◽  
Jutamas Ngampasutadol ◽  
Andrew D. Cox ◽  
Anna M. Blom ◽  
Lisa A. Lewis ◽  
...  
Keyword(s):  
Binding Protein ◽  
Alternative Pathway ◽  
Serum Resistance ◽  
Classical Pathway ◽  
Complement Pathway ◽  
Classical Complement Pathway ◽  
Pathway Activation ◽  
Normal Human ◽  
Classical Complement ◽  
Strain Background

ABSTRACT Lipooligosaccharide (LOS) heptose (Hep) glycan substitutions influence gonococcal serum resistance. Several gonococcal strains bind the classical complement pathway inhibitor, C4b-binding protein (C4BP), via their porin (Por) molecule to escape complement-dependent killing by normal human serum (NHS). We show that the proximal glucose (Glc) on HepI is required for C4BP binding to Por1B-bearing gonococcal strains MS11 and 1291 but not to FA19 (Por1A). The presence of only the proximal Glc on HepI (lgtE mutant) permitted maximal C4BP binding to MS11 but not to 1291. Replacing 1291 lgtE Por with MS11 Por increased C4BP binding to levels that paralleled MS11 lgtE, suggesting that replacement of the Por1B molecule dictated the effects of HepI glycans on C4BP binding. The remainder of the strain background did not affect C4BP binding; replacing the Por of strain F62 with MS11 Por (F62 PorMS11) and truncating HepI mirrored the findings in the MS11 background. C4BP binding correlated with resistance to killing by NHS in most instances. F62 PorMS11 and its lgtE mutant were sensitive to NHS despite binding C4BP, secondary to kinetically overwhelming classical pathway activation and possibly increased alternative pathway activation (measured by factor Bb binding) by the F62 background. FA19 lgtF (HepI unsubstituted) resisted killing by only 10% NHS, not 50% NHS, despite binding levels of C4BP similar to those of FA19 and FA19 lgtE (both resistant to 50% serum), suggesting a role for the proximal Glc in serum resistance independently of C4BP binding. This study provides mechanistic insights into how HepI LOS substitutions affect the serum resistance of N. gonorrhoeae.

scholarly journals THE DISTRIBUTION OF ANTIGENIC DETERMINANTS IN RAT SKIN COLLAGEN

1971 ◽  
Vol 133 (6) ◽  
pp. 1309-1324 ◽  
Author(s):  
Herbert Lindsley ◽  
Mart Mannik ◽  
Paul Bornstein
Keyword(s):  
Terminal Region ◽  
Antigenic Determinants ◽  
Collagen Molecule ◽  
Rat Skin ◽  
Amino Terminal ◽  
Skin Collagen ◽  
Native Collagen ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hyperimmune Rabbit

Immunological studies of rat skin collagen were carried out with a sensitive and quantitative radioimmunoassay. Hyperimmune rabbit antisera to rat skin collagen and isolated α2 chains were used. Iodine-labeled α chains and CNBr-produced peptides served as test antigens, and native collagen, α chains, and CNBr peptides were employed as inhibitors in the assay. The α1 and α2 chains were immunologically distinct. Although the α1 chain was not immunogenic, antibodies to α1 were detected in antisera to the intact collagen molecule. The major antigenic determinant of the α1 chain was located in α1-CB6 which constitutes the carboxy-terminal region of the chain. The α2 chain contained two non-cross-reacting antigenic determinants, one in the amino-terminal region (α2-CB1) and the other in the carboxy-terminal region (α2-CB5) of the chain. The native collagen molecule was less effective than isolated α chains in inhibiting binding of labeled peptides to antisera, indicating that antigenic determinants were less accessible in the triple helical molecule. These immunologic studies are consistent with preliminary comparative biochemical data which indicate that interspecies structural differences in collagen predominate at both the amino- and carboxy-terminal ends of the chains.

scholarly journals Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis

2017 ◽  
Vol 140 (1) ◽  
pp. 89-100.e2 ◽  
Author(s):  
Griet A. Van Roey ◽  
Christopher C. Vanison ◽  
Jeffanie Wu ◽  
Julia H. Huang ◽  
Lydia A. Suh ◽  
...  
Keyword(s):  
Chronic Rhinosinusitis ◽  
Complement Pathway ◽  
Classical Complement Pathway ◽  
Nasal Tissue ◽  
Pathway Activation ◽  
Classical Complement

scholarly journals The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

2005 ◽  
Vol 73 (4) ◽  
pp. 2400-2410 ◽  
Author(s):  
Ahmed S. Attia ◽  
Eric R. Lafontaine ◽  
Jo L. Latimer ◽  
Christoph Aebi ◽  
George A. Syrogiannopoulos ◽  
...  
Keyword(s):  
Human Serum ◽  
Resistant Strain ◽  
Moraxella Catarrhalis ◽  
Normal Human Serum ◽  
Serum Resistance ◽  
Complement Pathway ◽  
Classical Complement Pathway ◽  
Amino Acid Region ◽  
Normal Human ◽  
Classical Complement

ABSTRACT Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg2+ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-amino-acid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.

scholarly journals Antigenic and Immunogenic Investigation of B-Cell Epitopes in the Nucleocapsid Protein of Peste des Petits Ruminants Virus

2005 ◽  
Vol 12 (1) ◽  
pp. 114-121 ◽  
Author(s):  
Kang-Seuk Choi ◽  
Jin-Ju Nah ◽  
Young-Joon Ko ◽  
Shien-Young Kang ◽  
Kyoung-Jin Yoon ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peste Des Petits Ruminants ◽  
Serum Antibodies ◽  
N Protein ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Antigenic Domains ◽  
Carboxy Terminal

ABSTRACT Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.

scholarly journals Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

2001 ◽  
Vol 75 (23) ◽  
pp. 11851-11862 ◽  
Author(s):  
S. Michael Rothenberg ◽  
Mari N. Olsen ◽  
Louise Chang Laurent ◽  
Rachel Adams Crowley ◽  
Patrick O. Brown
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Receptor Binding Domain ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Env Protein ◽  
Carboxy Terminal ◽  
Murine Leukemia

ABSTRACT The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.

scholarly journals Regions of RNase E Important for 5′-End-Dependent RNA Cleavage and Autoregulated Synthesis

2000 ◽  
Vol 182 (9) ◽  
pp. 2468-2475 ◽  
Author(s):  
Xunqing Jiang ◽  
Alexis Diwa ◽  
Joel G. Belasco
Keyword(s):  
Intrinsic Property ◽  
General Characteristic ◽  
Rna Cleavage ◽  
Rnase E ◽  
Wild Type ◽  
E Coli ◽  
Amino Terminal ◽  
Catalytically Active ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

ABSTRACT RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5′ end. Here we show that the preference of E. coli RNase E for 5′-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived fromSynechocystis, indicating that the 5′-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5′-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. colicell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.

DEVELOPMENT OF HOMOLOGOUS IMMUNOLOGICAL ASSAYS FOR HUMAN PARATHYROID HORMONE

1980 ◽  
Vol 85 (1) ◽  
pp. 161-170 ◽  
Author(s):  
R. M. MANNING ◽  
G. N. HENDY ◽  
S. E. PAPAPOULOS ◽  
J. L. H. O'RIORDAN
Keyword(s):  
Parathyroid Hormone ◽  
Trichloroacetic Acid ◽  
Terminal Region ◽  
Human Parathyroid Hormone ◽  
Antibody Assay ◽  
Amino Terminal ◽  
High Affinity ◽  
Carboxy Terminal Region ◽  
Immunological Assays ◽  
Carboxy Terminal

SUMMARY Antisera to a trichloroacetic-acid precipitate of human parathyroid hormone (PTH) were produced in goats. Two of these antisera (G36 and G31) were of high affinity, and the bovine and porcine hormones were less reactive. Synthetic peptides containing the amino-terminal region of human PTH reacted with both antisera; the 1–34 peptide (PTH-(1–34)), with the sequence proposed by Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts in 1974, was more reactive than that having the sequence proposed by Brewer, Fairwell, Ronan, Sizemore & Arnaud in 1972. The antisera were further characterized with a number of other native and synthetic fragments of human PTH and reacted poorly with fragments from the carboxy-terminal region of the molecule. Since the amino-terminal fragments did not account for all the immunoreactivity, it is assumed that the antisera had some recognition sites for the central part of the molecule. Highly purified human PTH-(1–84) was labelled with 125I and radioimmunoassays were developed using this tracer and antiserum G36. To avoid the problems associated with labelling human PTH with 125I, a labelled antibody assay was developed with G36 and an immunoadsorbent consisting of human PTH-(1–34) (sequence of Niall et al.) coupled to cellulose. A sensitive homologous amino-terminal specific assay was developed in this way.

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