Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796216
Title: Isolation and characterisation of an inhibitor of complement mediated prevention of immune precipitation
Author: Ahmed, Alaa Eldin Elsayed
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1989
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
A normal plasma protein that inhibits complement- mediated prevention of immune precipitation (PIP) has been purified from normal human serum by three different methods- The first method was based on sequential affinity chromatography on IgG-Sepharose, protein A-Sepharose, and Con. A-Sepharose which resulted in the purification of a protein which was 60 kD on SDS-PAGE, 2. 9S on sucrose density gradient centrifugation, 58 kD molecular weight on gel filtration and had a pre-albumin electrophoretic mobility. The second method which was a modification of the first method involving the use of protease inhibitors during the purification procedure, produced a protein which was 60 kD molecular weight on SDS-PAGE, 19S on sucrose density gradient centrifugation, 1000 kD molecular weight by Sephacryl S-300 gel filtration and had a B2/g electrophoretic mobility. The third method did not involve an IgG-Sepharose affinity chromatography step but consisted of a combination of ion-exchange chromatography, gel filtration and heparin-Sepharose affinity chromatography with a final yield of 38% and 3360-fold purification. The final product of this purification procedure was a protein which shared the same characteristics with that prepared by method 2. A monospecific antiserum was produced by immunising rabbits with the protein purified by the first method. When tested in double-diffusion in agarose gel, the antiserum gave a reaction of complete identity with all three protein preparations. The present data suggest that the high molecular weight form of the protein is the polymer of the low molecular weight form. The purified protein stained positively in the PAS reaction, was sensitive to neuraminidase and trifluoro- methylsulphonic anhydride treatment, indicating that it is a glycoprotein. On account of its molecular weight on SDS- PAGE (60 kD), it has been called glycoprotein 60 (gp60). An ELISA procedure was developed using the anti-gp60 antiserum. The assay enabled me to measure gp60 levels in serum and to study the behaviour of serum gp60 on gel- filtration chromatography and sucrose density gradient centrifugation. Serum gp60 was found to be mainly in the high molecular weight form (19 S, 1000 kD), although a small proportion was of low molecular weight (2.9 S, 58 kD). Thus serum gp60 exists mainly as the polymer with a minor proportion being present as the monomer. In binding studies, [I]-gp60 was shown to bind to IgG but not to the IgA or IgM isotypes. The binding was localised to the Fc piece of IgG, and the subclasses IgG1 and IgG3 bound gp60 more effectively than did IgG2 or IgG4. Analysis of the data from the binding of [I]-gp60 to solid-phase BSA-IgG anti-BSA immune complexes (IC) showed that the affinity constant of gp60 for IgG was between 6.6 x10 and 11.2x10 1/mol for the monomer and 2.3x10 and 5.1x10 1/mol, and a single class of binding site was present. Saturation of binding was achieved when one molecule of gp60 monomer was bound for every five molecules of IgG, or when one molecule of gp60 polymer was bound for every 2k molecules of IgG. Further binding studies revealed that gp60 competed with Cl1, IgM-RF and F(abf) IgG-RF for binding to the Fc piece of IgG. Purified gp60 produced dose-dependent inhibition of PIP and solubilisation. PIP was the more sensitive to the effect of gp60. The polymeric form of gp60 inhibited PIP and solubilisation more effectively than the monomer. The mechanism of action of gp60 was studied by adding gp60 to normal serum and showing that it produced dose-dependent inhibition of IC-mediated activation of the classical pathway as shown by reduced formation of the C1s-C1 inhibitor complex, reduced consumption of C4a and C2 and reduced C4a and C3a generation. These data showed that gp60 prevents activation of the classical pathway by preventing Cl binding to IC. This leads to reduced C3 convertase formation and C3 cleavage so that the binding of C3b to IC is limited. Levels of gp60 in rheumatoid arthritis sera correlated with their ability to inhibit PIP. Gp60 which was present in normal serum was shown to regulate complement activation as the addition of Fab anti-gp60, but not normal rabbit IgG Fab, to sera resulted in 1) increased levels of haemolytic complement when EA(IgG) were used as targets, 2) increased complement activation by IC and 3) increased levels of PIP activity. It was concluded that in normal serum gp60 regulated complement activation. The identity of gp60 is unknown. It was shown to be distinct from the proteins recognised by 57 antisera. So far attempts at amino acid sequencing have failed, and cDNA expression libraries are being screened to identify positive cDNA clones which can be used for nucleotide sequencing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796216  DOI: Not available
Share: