Biomedical subjects

T C Farries

Publications and source records attributed to T C Farries.

13 recordsLinked to original sources

PubMed1991

Removal of N-acetyl groups from blocked peptides with acylpeptide hydrolase. Stabilization of the enzyme and its application to protein sequencing.

Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then absorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.

Amino Acid Sequence↗

PubMed1991

Enzyme-mediated peptide synthesis using acylpeptide hydrolase.

Acylpeptide hydrolase is shown to catalyse the specific addition of a single amino acid to the N-terminus of a peptide. The stabilised Sepharose-coupled form of the enzyme is used to couple a carboxy-methylated N-formyl (or N-acetyl) amino acid to a short pre-existing peptide. The yield is improved by optimal timing of the reaction and the presence of moderate concentrations (5%) of N,N-dimethylformamide. Two tripeptides, Ac-Ala-Ala-Ala and fMet-Leu-Phe (f, formyl) were synthesized by this technique (in yields of 2% and 0.064% respectively). The products were characterised by HPLC, amino acid analysis, mass spectroscopy and protein sequencing. The synthetic fMet-Leu-Phe also had biological activity, in that it stimulated superoxide generation by granulocytes. Acylpeptide hydrolase could therefore be a very useful tool for the synthesis and modification of peptides.

Amino Acids↗

PubMed1991

Evolution of the complement system.

The ancestral form of the alternative pathway of complement activation probably originated as a primitive independent immune system. Subsequent evolution of an adaptive immune response drove the specialization of the classical pathway to connect antibody-mediated nonself recognition to the complement-dependent effector mechanisms. In this article Timothy Farries and John Atkinson consider how the contemporary complexity arose by a succession of credible alterations at the genetic level, and the selective advantages provided at each step.

Biological Evolution↗

PubMed1990

The mechanism of activation of the alternative pathway of complement by cell-bound C4b.

Investigations into the mechanism of alternative pathway-dependent lysis of C4b-coated cells are reported. Test cells (EAC1q4b) were formed by reaction of sheep erythrocytes with antibody, C1 and C4. In C5-deficient serum, more C3b was deposited onto EAC1qC4b than onto control cells (EAC1q). The possibility that the C4bBb enzyme could form was considered, but no C3 convertase activity was generated when magnesium, properdin and factors B and D were added to EAC1qC4b. Binding studies employing radiolabeled components provided evidence that C4b bound the C3 convertase, C3bBbP, through a weak interaction with C3b. These data implied C3 conversion would be localized to the cell surface, thereby amplifying C3b deposition. This could be demonstrated in vitro. C3b, properdin, factor B and factor D were all required and the amplified C3b deposition was not due to deposition onto C4b itself. In serum, C5 convertase activity would be consequently expressed and cell lysis would result. This could be the mechanism by which the sera of C2-deficient patients mediate lysis of antibody coated sheep erythrocytes.

Animals↗

PubMed1990

Evolutionary implications of a new bypass activation pathway of the complement system.

The classical pathway of complement activation is a highly specific and amplifiable effector system responding to recognition of foreign antigens by antibody. It comprises a group of well characterized proteins in mammalian plasma. There are many similarities with the alternative pathway of complement activation, which suggests that they have a common evolutionary origin. Both pathways have homologous components, use related activation and regulatory mechanisms, result in the release of the anaphylatoxins C3a and C5a, and deposit C3b onto activating surfaces. This fixed C3b then becomes the focus of further immune reactions, involving either the lytic complement components or C3b receptors on effector cells. Phylogenetic data indicate that the alternative pathway is the older, and that the classical pathway evolved from it. Here Timothy Farries and colleagues review this evolutionary process and present a possible sequence of events that is suggested by recent functional data from their laboratory.

Animals↗

PubMed1990

Competition for binding sites on C3b by CR1, CR2, MCP, factor B and factor H.

The reaction of radiolabeled C3b-binding proteins with C3b-coated particles has been investigated. CR1 binding was inhibited by factor H and factor B (in the presence of properdin), but not by properdin alone. CR2 and MCP binding were also inhibited by factor H. Therefore factor H, factor B, CR1, CR2 and MCP probably comprise a group of mutually competitive proteins with similar or overlapping binding sites on C3b. These results correlate with their structural homology and suggest that they all evolved from a single C3b-binding molecule. Factor H, CR1 and MCP are also cofactors for the factor-I-mediated cleavage of C3b. A species incompatibility between rat factor I and human CR1 for the cleavage of human C3b suggests the possibility that cofactors may also function by interacting directly with factor I.

Animals↗

PubMed1989

Lysis of sensitized sheep erythrocytes in human sera deficient in the second component of complement.

Analysis of C-dependent lysis of sensitized SRBC by C2-deficient sera (C2D) led to the characterization of a C2 bypass pathway. Lysis in the total hemolytic C assay by C2D sera was Ca2+-dependent and required a high concentration of hemolysin to sensitize E. Selective component depletion indicated a requirement for C1 and C4 of the classical pathway (CP) and proteins B, P, and probably D of the alternative pathway (AP). Total hemolytic C could be restored to normal in these C2D sera by utilizing heavily sensitized E or by the addition of a supranormal concentration of B. This system most closely resembles a pathway described by J. E. May and M. M. Frank which requires antibody, C1, and the AP but not C4 or C2. It differs in its requirement for C4. We hypothesize that this pathway represents vestiges of a more primitive C pathway. It becomes evident and possibly clinically important in the setting of C2 deficiency, by allowing C activation, other than the AP, and perhaps in normal individuals, by damaging microorganisms that have evolved means to inhibit early components of the CP.

Adult↗

PubMed1989

Biosynthesis of properdin.

Properdin (P) is synthesized by the human promyelocytic cell line, HL-60, after differentiation with DMSO. The secreted P was physiochemically indistinguishable from purified plasma P. It was polymerized and able to bind to C3IBb-Sepharose but not to C3i-Sepharose. No extracellular precursor was present. The intracellular form, detected between 1 and 4 h after labeling, was similar but had a slightly lower Mr. It also bound reversibly to C3iBb-Sepharose, and polymers could be demonstrated by cross-linking. Pulse-chase experiments suggested the existence of an earlier, but undetectable, intracellular precursor(s). This form could not be immunoprecipitated even when harsh solubilization conditions and/or antibodies against reduced and denatured P were utilized. Studies with endoglycosidases F and H and tunicamycin indicated that the detectable intracellular precursor contains high mannose N-linked carbohydrate that is processed to the complex form before secretion. The sugars are not required for polymerization, secretion, or functional activity, or responsible for the electrophoretic heterogeneity. Polymerization of P is therefore an early intracellular event, perhaps carefully controlled to prevent anomalous aggregation.

Carbohydrate Conformation↗

PubMed1989

Sulfation of tyrosine residues increases activity of the fourth component of complement.

Sulfation of tyrosine residues recently has been recognized as a biosynthetic modification of many plasma proteins and other secretory proteins. Effects of this site-specific modification on protein function are not known, but the activity of several peptides such as cholecystokinin is greatly augmented by sulfation. Here, we examine the role of sulfation in the processing and activity of C4 (the fourth component of complement), one of the few proteins in which sites and stoichiometry of tyrosine sulfation have been characterized. Our results, with C4 as a paradigm, suggest that sulfation of tyrosine residues can have major effects on the activity of proteins participating in protein-protein interactions. Sulfation of C4 synthesized by Hep G2 cells was blocked by incubating the cells with NaClO3 and guaiacol. These sulfation inhibitors did not alter secretion or other steps in the processing of C4. However, hemolytic activity of C4 was decreased more than 50%. The inhibitors' effect on C4 activity was prevented by adding Na2SO4 to restore sulfation of C4. Activity of C3, a complement component homologous to C4 but lacking tyrosine sulfate residues, was minimally reduced (19%) by the inhibitors. Decreased hemolytic activity of nonsulfated C4 apparently resulted from impaired interaction with complement subcomponent C1s (EC 3.4.21.42), the protease that physiologically activates C4. Purified C1s was able to cleave nonsulfated C4, but approximately 10-fold higher concentrations of C1s were required for that cleavage than to yield equivalent cleavage of sulfated C4. Our results suggest that activation of C4, a central component in the classical pathway of complement activation, is influenced by the level of sulfation of the protein. Thus, sulfation of C4 provides a potential locus for physiological or pharmacological modulation of complement-mediated opsonization and inflammation.

Cell Line↗

PubMed1988

Analysis of the interaction between properdin and factor B, components of the alternative-pathway C3 convertase of complement.

The interactions between Factor B (B), its activation products Ba and Bb, properdin (P) and C3i or C3b, components that together form the alternative-pathway C3 convertase enzyme of human complement, have been analysed. Fluid-phase complexes of the purified components C3i, B and P were probed with the homobifunctional cross-linking reagent disuccinimidyl tartarate, and efficient cross-linking of B to P was observed. The 140 kDa B-P conjugate formed was cleaved by Factor D to yield a single product of 85 kDa. This is consistent with a Ba-P heterodimer, and suggests that the initial interaction of B and P includes an interaction of P with the Ba domain of intact B. (The Ba fragment is not retained in the active P-stabilized complex, C3bBbP). By contrast, no cross-linking of P to the Bb domain of B could be demonstrated. Binding studies on cellular intermediates also provided evidence for a site of interaction between B and P, with high concentrations of B inhibiting P binding to EAC3b (sheep erythrocytes coated with antibody and C3b). Neither isolated Ba nor Bb had any effect on the P-EAC3b interaction. High concentrations of B also accelerated the decay of the functional EAC3bBbP complex. These data indicate that the positive co-operativity of binding to C3i or to C3b between B and P is mediated, at least in part, through a direct interaction between B and P.

Complement Activating Enzymes↗

PubMed1988

Analysis of the interactions between properdin, the third component of complement (C3), and its physiological activation products.

The interactions of properdin with both surface-bound and fluid-phase C3 (the third component of complement) and its activation products have been investigated by using a purified preparation of the 'native' form. At physiological ionic strength, a weak interaction with cell-bound C3b (the larger activation fragment of C3) could be demonstrated. In the presence of Factor B this interaction was enhanced, and further enhancement was seen when C3bBb sites were formed on the erythrocytes. The avidities of properdin for cell-bound iC3b (the initial product of Factors I and H action on C3b) and C3b were compared at low ionic strength, with that measured for iC3b being less than that for C3b. In contrast, the affinities of properdin for fluid-phase C3b, iC3b and C3c (the larger product of Factors I and H or CR1 (the C3b receptor) action on iC3b) were all very similar, and apparently much weaker than that for cell-bound C3b. No interaction with either native C3 or, more surprisingly, C3i (haemolytically inactive C3) could be detected. Properdin also inhibited Factor I binding to, and action upon, cell-bound C3b, but did not inhibit Factor I action on fluid-phase C3b. These data permit a more detailed description of the roles of properdin in the alternative pathway of complement activation, emphasizing its importance in concentrating activation at the activating surface.

Complement Activation↗

PubMed1988

Structure of C3f, a small peptide specifically released during inactivation of the third component of complement.

C3f, a peptide presumed to be generated by the combined actions of factors I and H on fluid-phase C3b, has been isolated and sequenced. The peptide is 17 residues long and has a molecular weight of 1,847 daltons. The amino-terminal sequence is, with the exception of a single residue, identical to that deduced for the 46-kilodalton polypeptide seen transiently in the generation of iC3b from C3b, and is in full agreement with the sequence deduced from cDNA analysis. In addition, high-pressure liquid chromatography of the digestion of C3b by factor I has shown that C3f is the sole peptide released during iC3b generation.

Amino Acid Sequence↗

PubMed1987

Resolution and analysis of 'native' and 'activated' properdin.

A rapid and reproducible procedure for the resolution of 'native' and 'activated' forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported. This fractionation permitted effective screening of samples for conditions that cause activation. Only 'native' properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls. Transformation of 'native' into 'activated' properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb. Electron microscopy showed that only the 'native' population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Müller-Eberhard [(1984) J. Biol. Chem. 259, 4582-4588]. 'Activated' properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates. We therefore conclude that properdin 'activation' is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation. Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the 'native' population.

Cell Wall↗