Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis

Table 1 Clinical, biochemical, histological and serological data in 95 patients with PBC (mean ± SD)

Parameters
Females:males	88:7
Age (yr)
mean ± SD	50 ± 9.8
Range	21-70
Biochemical parameters1
AP (U/L) (normal < 120)	453 ± 451
GT (U/L) (normal < 50)	154 ± 143
ASAT (U/L) (normal < 35)	39.4 ± 42.6
ALAT (U/L) (normal < 35)	51 ± 49.3
Cholinesterase (kU/L) (normal > 5)	4.7 ± 1.7
Bilirubin (mg%) (normal < 1.5)	1.62 ± 0.8
Cholesterol (mg/dL) (normal < 200)	251 ± 69
Eosinophils (%) (normal < 4)	3.61 ± 2.36
IgG globulins (mg%) (normal < 1800)	1679 ± 855
IgA globulins (mg%) (normal < 400)	269 ± 158
IgM globulins (mg%) (normal < 280)	416 ± 255
Histological data2
PBC stage I/II (n)	45
PBC stage III/IV (n)	20
Serological data
Antimitochondrial antibodies (number positive)
AMA (IFT) (titer > 1:160)	95
Anti-M2 (ELISA)
Total	95
IgG type	87
IgM type	72
Anti-PDC-E2 (ELISA)
Total	95
Antinuclear antibodies (number positive) (titer > 1:160)
Nuclear dots (sp 100)	16
Nuclear membrane (gp 210)	10
Centromeres	8

¹Referring only to the 65 untreated patients;

²Liver biopsy had been performed in 65 patients; 42 of them were untreated, and of these patients 30 were in stage I/II and 12 in stage III/IV. IFT: Immunofluorescence test; ELISA: Enzyme-linked immunosorbent assay; PBC: Primary biliary cirrhosis; PDC-E2: Pyruvate dehydrogenase complex E2-component.

Table 2 Reactivity and incidence of IgG-antibodies to 33 PDC-E2 peptides in PBC patients as compared to healthy individuals n (%)

	Peptide No.	Amino acid	PBC patients		Healthy individuals (n = 22)
			Anti-M2+ (n = 95)	AMA-/ANA+ (n = 11)
Outer lipoyl domain (aa 1-80)	1	-2-23	4 (4)	0	1 (5)
	2	16-40	6 (6)	0	2 (9)
	3	33-57	10 (11)	0	2 (9)
	4	50-74	15 (16)	0	0
	5	67-91	26 (27)	0	2 (9)
Hinge	6	84-108	17 (18)	0	3 (14)
	7	101-125	42 (44)1	0	0
Inner lipoyl domain (aa 133-217)	8	118-142	14 (15)	0	1 (5)
	9	135-159	32 (34)	0	0
	10	152-176	28 (29)	0	2 (9)
	11	169-193	11 (12)2	0	2 (9)
	12	186-210	16 (17)	0	1 (5)
	13	203-227	6 (6)	0	3 (14)
Hinge	14	220-244	4 (4)	0	2 (9)
	15	237-261	3 (3)	0	4 (18)
	16	254-278	20 (21)	0	1 (5)
E1/E3-binding site (aa 272-303)	17	271-295	3 (3)	0	1 (5)
	18	288-312	7 (7)	0	1 (5)
Hinge	19	305-329	19 (20)	0	3 (14)
Catalytic site (aa 331-561)	20	322-346	12 (13)	2 (18)	1 (5)
	21	339-363	0	0	1 (5)
	22	356-380	19 (20)	0	1 (5)
	23	373-397	2 (2)	1 (9)	1 (5)
	24	390-414	1 (1)	1 (9)	2 (9)
	25	407-431	53 (56)3	0	4 (18)
	26	424-448	10 (11)	0	1 (5)
	27	441-465	5 (5)	0	2 (9)
	28	458-482	18 (19)	0	0
	29	475-499	55 (58)4	0	1 (5)
	30	492-516	9 (9)	0	0
	31	509-533	17 (18)	0	2 (9)
	32	526-550	2 (2)	0	1 (5)
	33	543-561	11 (12)	0	1 (5)

¹Twelve patients had antibodies only of the IgG-, 28 only of the IgM-, and 30 of the IgG- and IgM-type; in total, 70 (71%) reacted with peptide 7;

²Four patients had antibodies only of the IgG-, 30 only of the IgM-, and 7 of the IgG- and IgM-type; in total, 41 (43%) reacted with peptide 11;

³Seventeen patients had antibodies only of the IgG-, 18 only of the IgM-, and 37 of the IgG- and IgM-type; in total, 72 (76%) reacted with peptide 25;

⁴Two had antibodies only of the IgG-, 16 only of the IgM-, and 53 of the IgG- and IgM-type; in total, 71 (75%) reacted with peptide 29. Similar data as for healthy controls were obtained with sera from patients with auto-immune hepatitis, alcoholic liver disease and collagen disorders. Peptides 3, 10, 11, 25 and 29 are previous and novel immunodominant epitopes. +: Positive; -: Negative.

Table 3 Reactivity and incidence of IgM-antibodies to 33 PDC-E2 peptides in PBC patients as compared to healthy individuals n (%)

	Peptide No.	Amino acid	PBC patients		Healthy individuals (n = 22)
			Anti-M2+ (n = 95)	AMA-/ANA+ (n = 11)
Outer lipoyl domain (aa 1-80)	1	-2-23	7 (7)	0	1 (5)
	2	16-40	10 (11)	0	2 (9)
	3	33-57	27 (28)	0	0
	4	50-74	33 (35)	1 (9)	1 (5)
	5	67-91	42 (44)	0	1 (5)
Hinge	6	84-108	31 (33)	0	1 (5)
	7	101-125	52 (55)1	1 (9)	2 (9)
Inner lipoyl domain (aa 133-217)	8	118-142	34 (36)	0	1 (5)
	9	135-159	35 (37)	0	2 (9)
	10	152-176	35 (37)	0	1 (5)
	11	169-193	37 (39)2	0	2 (9)
	12	186-210	27 (28)	0	1 (5)
	13	203-227	23 (24)	0	2 (9)
Hinge	14	220-244	26 (27)	0	0
	15	237-261	43 (45)	0	3 (14)
	16	254-278	33 (35)	0	1 (5)
E1/E3-binding site (aa 272-303)	17	271-295	15 (16)	0	0
	18	288-312	24 (25)	0	1 (5)
Hinge	19	305-329	36 (38)	0	3 (14)
Catalytic site (aa 331-561)	20	322-346	1 (1)	0	0
	21	339-363	4 (4)	0	0
	22	356-380	24 (25)	0	1 (5)
	23	373-397	10 (11)	0	0
	24	390-414	23 (24)	1 (9)	3 (14)
	25	407-431	55 (58)3	0	1 (5)
	26	424-448	21 (22)	0	0
	27	441-465	16 (17)	0	0
	28	458-482	15 (16)	0	0
	29	475-499	70 (74)4	0	1 (5)
	30	492-516	14 (15)	0	2 (9)
	31	509-533	6 (6)	0	2 (9)
	32	526-550	4 (4)	0	0
	33	543-561	9 (9)	0	1 (5)

^1,2,3,4Same as Table 2. Peptides 3, 7, 10, 11, 25 and 29 are previous and novel immunodominant epitopes.

Table 4 Reactivity of sera from 95 anti-M2 positive PBC patients with different peptide conjugates containing the reported immunodominant epitope of PDC-E2[6,12]

Peptides of the inner lipoyl domain	IgG-antibodies			IgM-antibodies
	Positive n (%)	mean absorbance × 1000 ± SD (median)	Cut off value	Positive n (%)	mean absorbance × 1000 ± SD (median)	Cut off value
152-176 (present study peptide 10)	28 (29)	153 ± 243 (87)	150	35 (37)	95 ± 77 (73)	80
169-193 (present study peptide 11)	11 (12)	210 ± 219 (157)	300	37 (39)	143 ± 120 (101)	120
167-184	0	113 ± 63 (93)	100	5 (5)	53 ± 79 (20)	100
167-184-LA	3 (3)	135 ± 71 (111)	100	10 (11)	78 ± 107 (40)	100
OVA-167-1841	42 (44)	316 ± 260 (271)	100	54 (57)	254 ± 318 (117)	100
OVA-167-184-LA1	21 (22)	175 ± 280 (148)	100	29 (31)	113 ± 217 (52)	100

¹After subtraction of reactivity to OVA alone.