Xenobiotics and loss of tolerance in primary biliary cholangitis

Table 1 Serological reactivity of primary biliary cholangitis sera to the recombinant proteins of wild type pyruvate dehydrogenase E2 lipoyl domain, single amino acid mutants double, triple and quadruple mutants¹

Mutant No.		Amino acid sequence	PBC sera2		Purified PBC IgG3
			IgG	IgM
PDC-E2 wild type		LLAEIETDKATIGFEVQEE	1	1	1
Mutant 3		LLAEAETDKATIGFEVQEE	0.476 ± 0.029	0.504 ± 0.043	0.408 ± 0.052
Mutant 9		LLAEIETDKATAGFEVQEE	0.706 ± 0.029	0.781 ± 0.054	0.552 ± 0.065
Mutant 12		LLAEIETDKATIGFAVQEE	0.659 ± 0.034	0.768 ± 0.096	0.482 ± 0.074
Double amino acid substitution	Mutant 1	ALAEIETDKATAGFEVQEE	0.334 ± 0.029	0.253 ± 0.034	0.075 ± 0.023
	Mutant 2	ALAEIETDKATIGFAVQEE	0.461 ± 0.031	0.435 ± 0.045	0.663 ± 0.069
	Mutant 3	LLAEAETDKATAGFEVQEE	0.066 ± 0.009	0.093 ± 0.016	0.024 ± 0.007
	Mutant 4	LLAEAETDKATIGFAVQEE	0.111 ± 0.017	0.095 ± 0.016	0.043 ± 0.016
Triple amino acid substitution	Mutant 1	ALAEAETDKATAGFEVQEE	0.017 ± 0.004	0.044 ± 0.009	0.038 ± 0.017
	Mutant 2	LLAEAETDKATAGFAVQEE	0.019 ± 0.003	0.054 ± 0.012	0.050 ± 0.007
Quadruple amino acid substitution		ALAEAETDKATAGFAVQEE	0.024 ± 0.005	0.066 ± 0.012	0.075 ± 0.031

¹16 single alanine substitution mutants along a peptide that constitutes the beta sheet of the PDC-E2 inner lipoyl domain, 4 double aa substitution mutants, 3 triple and one quadruple mutants were also constructed. Purified proteins from all these constructs were analyzed for Ig reactivity with PBC sera. 3/16 of the single amino acid mutants have much reduced antibody binding are shown. Other alanine substitutions have Ig reactivity similar to wild type PDC-E2. Control sera samples include (lupus, n = 30, Crohn’s disease, n = 20, PSC, n = 28, scleroderma n = 20) did not react;

²Relative ratio of serological IgG and IgM reactivity compared to wild type determined by ELISA at 1:4000 sera dilution (n = 60);

³Relative ratio of purified IgG reactivity to wild type determined by ELISA (n = 10). PBC: Primary biliary cholangitis.