Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence

Smith, Michael A.; Gerrie, Linda M.; Dunbar, Bryan; Fothergill, John E.

doi:10.1042/bj2070253

Cited by 33 publications

(26 citation statements)

References 29 publications

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“…In fact, a Lys-Lys-Lys-Ile-Glu-GluGlu sequence is found at the N-terminus from the third to the ninth residue, and in the region between Arg-62 and Lys-68 there is only one residue which is uncharged at neutral pH. A comparison of the sequence of bovine C5a with that of other anaphylatoxins indicated a homology of 42% with bovine C4a [38] and of 32% with human C3a [35]. On the contrary, a computer search of homologies with other known protein sequences provided minor scores.…”

Section: Discussionmentioning

confidence: 99%

C5a fragment of bovine complement

Gennaro

Simonic²,

Negri³

et al. 1986

European Journal of Biochemistry

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1. C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. 2. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. 3. After preparing a rabbit antiserum to bovine CSa/des-Arg-CSa, a competitive enzyme-linked immuno sorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 pg/ml. 4. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. 5. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. 6 . Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of a-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.Chemotaxis of inflammatory cells may be induced by products of humoral and cellular immune or immune-related reactions, and by products elaborated by bacteria or by the inflammatory cells themselves [l]. Among these proinflammatory agents the complement fragment C5a is one of the most potent, also eliciting generation of cytotoxic oxygen radicals and secretion of granule content from both neutrophils and macrophages [2-101. Furthermore, this polypeptide exhibits classical anaphylatoxic properties, i.e. it causes smooth muscle contraction, histamine release from mast cells and enhanced vascular permeability [I, 2, 11 -131.A clear picture of the relation between structure and function of this chemotactic and spasmogenic molecule, which consists of 74 amino acid residues, is not yet available [8]. In fact, (a) there is paucity of sequence-homology information, because only the primary structures of human and porcine C5a have been elucidated [14-161, and (b) scarcity of suitable quantities of C5a has hampered the determination of its secondary and tertiary structure by physico-chemical techniques.Starting from several liters of bovine serum, we have purified milligram amounts of C5a and des-Arg-CSa, whose effect on neutrophils have then been compared. The amino acid

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Section: Discussionmentioning

confidence: 99%

C5a fragment of bovine complement

Gennaro

Simonic²,

Negri³

et al. 1986

European Journal of Biochemistry

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“…Unlike C3a and C5a, C4a appears to have little, if any, activity in humans although C4a has been reported to be a major mediator in inner ear damage (Harada et al, 1992). The amino acid sequences of C4a obtained to date-such as rat (Cui et al, 1988), human (Moon et al, 1981), and bovine (Smith et al, 1982)-show conservation of the 6 Cys residues likely to form the disulfide knot and the basic residues that are present in C3a and C5a (Fig. 2B).…”

Section: Structure Of Complement Peptidesmentioning

confidence: 99%

International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors

Klos

Wende²,

Wareham³

et al. 2013

Pharmacol Rev

224

245

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“…The explanation for the three original CNBr-cleavage peptide pools must be that the CNBr cleavage of the intact arom enzyme was incomplete, E. coli peptide. The peptide EC4 was sequenced by automated gas-phase sequencing on a Beckman model 890 liquid-phase sequencer as described previously (Smith et al, 1982). The radioactivity released at each cycle was determined by liquidscintillation counting.…”

Section: Digestion With Cnbrmentioning

confidence: 99%

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

Chaudhuri

Duncan

Graham

et al. 1991

Biochemical Journal

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The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.

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Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence

Cited by 33 publications

References 29 publications

C5a fragment of bovine complement

C5a fragment of bovine complement

International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

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