A serum sweet-doughnut protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis

Transcription

1 Supplementary information A serum sweet-doughnut protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis Atsushi Kuno 1#, Yuzuru Ikehara 1#, Yasuhito Tanaka, Kiyoaki Ito 3, Atsushi Matsuda 1, Satoru Sekiya 1, Shuhei Hige, Michiie Sakamoto 5, Masayoshi Kage 6, Masashi Mizokami 3 and Hisashi Narimatsu 1* 1 Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan; Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 3 The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan; Department of Internal Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 5 Department of Pathology, School of Medicine, Keio University, Japan; 6 Department of Pathology, Kurume University School of Medicine, Kurume, Japan #These authors contributed equally to this study. *Contact Information: Hisashi Narimatsu, Ph.D., M.D. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Umezono, Tsukuba, Ibaraki , Japan. Tel: h.narimatsu@aist.go.jp

2 1. Blood sampling. On-site assay (<30 min) PLT counting Biochemical assay New assay 3. Inquiry Supplementary Fig.1.

3 HV HCC HV HCC NC HV HCC HV HCC Sialidase A - + N-Glycosidase F - + Bio-IgG HV: healthy volunteer with normal livers HCC: patient with hepatocellular carcinoma NC: negative control Bio-IgG: capturing antibody for immunoprecipitation Supplementary Fig..

4 a. Ab Ab sandwich immunoassay for total MBP detection b. Lectin Ab sandwich immunoassay for aberrant MBP detection : antibody against protein moiety of MBP : lectin with specific affinity to fibrosis-related glycan : Strong affinity (K d <10 9 M) applicable for rapid assay : Weak affinity (K d =10 3 ~10 6 M) Capturing probe (immobilized on plate) Antibody Lectin Antibody Detecting probe (overlaid) Antibody Antibody Lectin Specific binding to aberrant MBP Low High High Sensitive detection High High Low This study Supplementary Fig. 3.

5 5 lectins in LecChip Step1: Glycan profiling of serum MBP by ALP (15 patients with HCV) 7 lectins binding to serum MBPs PSA, LCA, AOL, AAL, MAL, SNA, SSA, TJAI, PHAL, ECA, RCA10, PHAE, DSA, NPA, ConA, GNA, HHL, BPL, TJAII, LEL, ABA, STL, UDA, PWM, WFA, Jacalin, WGA Step: Subtraction of high-noise lectins based on the lectin microarray analysis of abundant serum glycoproteins 6 lectins with high fidelity MAL, ECA, BPL, TJAII, PWM, WFA Step3: Selection of the best lectin by statistical analysis of the data obtained in Step1 (ROC & Mann-Whitney) 1 lectin with highest accuracy WFA Verification of lectin-antibody sandwich ELISA for direct measurement of target proteins in serum Supplementary Fig..

6 High-noise lectin PSA LCA AOL AAL MAL SNA SSA TJA-I PHA-L ECA RCA10 PHA-E DSA NPA ConA GNA HHL BPL TJA-II ABA LEL STL UDA PSA AOL MAL SSA PHA-L RCA10 DSA ConA HHL TJA-II LEL UDA PWM Jacalin WFA WGA Jacalin WGA Net intensity PSA LCA AOL AAL MAL SNA SSA TJA-I PHA-L ECA RCA10 PHA-E DSA NPA ConA GNA HHL BPL TJA-II ABA LEL STL UDA PWM Jacalin WFA WGA Net intensity a. [Lectin] MBP : MBP averaged in 15 patients b. [Lectin] Serum : HV pool serum Lectin F = log ([Lectin] MBP / [Lectin] Serum ) Image Graph representation (Fig. b) High-S/N lectin Black box: high-noise lectins Red box: high signal-to-noise (S/N) lectins log ([Lectin] MBP / [Lectin] Serum ) Supplementary Fig. 5.

7 TJAII Sensitivity Sensitivity Sensitivity Sensitivity Sensitivity Relative intensity (DSA=1.0) PWM Relative intensity (DSA=1.0) WFA MAL Relative intensity (DSA=1.0) ECA Relative intensity (DSA=1.0) BPL.5 0. MAL (r=0.9) ECA (r=0.55) BPL (r=0.58) F01 F F3 F STAGING Fibrosis score F01 F F3 F STAGING Fibrosis score F01 F F3 F STAGING Fibrosis score TJAII (r=0.59) PWM (r=0.50) WFA (r=0.56) 108 Relative intensity (DSA=1.0) Relative intensity (DSA=1.0) F01 F F3 F STAGING Fibrosis score F01 F F3 F STAGING Fibrosis score F01 F F3 F STAGING Fibrosis score Cirrhosis Severe fibrosis Significant fibrosis WFA PWM TJAII BPL HHL WFA PWM TJAII BPL HHL WFA PWM TJAII BPL HHL MAL ECA HHL BPL TJAII PWM WFA ECA ECA ECA Specificity MAL MAL Specificity 1-Specificity MAL Supplementary Fig. 6.

8 WFA-agarose Affinity capturing IP WB HCC1 HCC HCC3 LC1 M T E T E M T E T E PC LC LC3 HV1 HV M T E T E M T E T E PC M: molecular weight marker T: path-through fraction in WFA-agarose chromatography E: elution fraction in WFA-agarose chromatography HV: healthy volunteer with normal livers HCC: patient with hepatocellular carcinoma LC: patient with liver cirrhosis PC: positive control which is MBP immunoprecipitated from commercial serum of a healthy volunteer. Supplementary Fig. 7.

9 Absorbance at 00 nm Absorbance at 00 nm a. Recombinant human MBP 1. b. Human MBP / HepG Native Heat-denature [rhmbp] mg/ml [Total protein] mg/well Supplementary Fig. 8.

10 Relative intensity (DSA = 1.0) Absorbance at 00 nm a. Direct measurement by sandwich ELISA HCC1 HCC HCC3 LC1 LC8 LC LC9 LC3 LC10 HV1 GP1 HV GP b. IP antibody-overlay lectin microarray HCC1 HCC HCC3 LC1 LC LC3 HV1 HV HV: healthy volunteer with normal livers HCC: patient with hepatocellular carcinoma LC: patient with liver cirrhosis Supplementary Fig. 9.

11 Nagoya City Univ. Chronic hepatitis patients n=15 (F01=33, F=3, F3=31, F=9) Healthy volunteers (HV) n=70 i) Sample recruitment Hokkaido Univ. Chronic hepatitis patients n=8 (F01=33, F=3, F3=31, F=9) AIST Healthy volunteers (HV1) n=8 ii) HISCL measurement AIST 9 patients and 118 healthy volunteers with missing data in ALT, AST, hyaluronic acid iii) Statistical analysis AIST Chronic hepatitis patients n=160 (F01=66, F=1, F3=33, F=0) Supplementary Fig. 10.

12 HV3 HV1 HV F01 F F3 F N Minimum 8,33 30,97 33,89 5,773 55,869 8, ,88 5 th percentile 30,91 56,60 6,33 80,103 1,181 57, ,1 Median 5,18 69,93 81,19 13,807 09,53 37,10 1,37, th percentile 6,05 99, ,617 38,63,07 705,91,510,981 Maximum 5,871 3,787 37,77 1,63,710 1,687,889,695,70 6,73,933 Mean 53,71 79,535 9,165 05, ,67 575,089 1,67,515 SD 38,515 3,377 50,560 17,888 36,557 5,35 1,31,776 SE 1,18,96 6,03,06 50,78 8,60 1,900 HV3 (1000) HV1 (7) HV (70) F01 (8) F (5) F3 (0) F (35) Lower 95% CI of mean 51,351 69,553 80, ,836 55,736 08,008 1,3,558 Higher 95% CI of mean 56,131 89,517 10,1 53,587 57,611 7,171,15,7 Horizontal lines inside each box represent the median, and the lower and upper borders of the box encompass the interquartile range. Vertical lines from the ends of each box encompass the 5 th and 95 th percentiles. Supplementary Fig. 11.

13 Chemiluminescent intensity Chemiluminescent intensity FastLec-Hepa 10 7 Activity 10 7 Fibrosis A0 (9) A1 (65) A (6) Grade A3 (7) F01 (7) F (7) F3 (31) Stage F (10) Scatterplots of the data obtained with FastLec-Hepa against the hepatic activity (grade: A0-A3) and the fibrosis score (stage: F0- F). Red horizontal lines represent the median. Data of HCV patients (n=16) available with both grade and stage defined by HAI among the participants (n=09). Supplementary Fig. 1.

14 FastLec-Hepa ([Post] [Pre]) 500, ,000-1,000, ALT ([Post] [Pre]) Scattered plots of changes in the ALT levels and FastLec-Hepa counts in patients with sustained virologic response (SVR) and relapse/nonresponders (non-svr) during interferon therapy. Supplementary Fig. 13.

15 Chemiluminescent intensity by X10 dilution measurement 1.5E E E+05 y = 0.095x R² = E E E E E+07 Chemiluminescent intensity by direct measurement HCV patients and healthy volunteers participated in this experiment was also used in Supplementary Fig.10. Supplementary Fig. 1.

16 Supplementary Table 1.

17 Supplementary Table 1.

18 Supplementary Table 1.

19 Supplementary Table 1.

20 Median Mean±SD (95% CI lower - higher) All (F0-) F01 F F3 F Patient number (N) Age (years) ±1.7 ( ) ±13.5 ( ) ±1.8 ( ) ±9.6 ( ) ALT (IU/L) AST (IU/L) Platelets (10 9 /L) Hyalurinic acid (ng/ml) AGP: AOL/DSA AGP: MAL/DSA FastLec-Hepa (X10 5 ) ±136.6 ( ).5 6.7±70.0 ( ) ±59.9 ( ) ±771 ( ).3 8.0±18.0 ( ) ±3.1 ( ). 5.±8.1 (.0-6.5) ±0.9 ( ) ±95.8 ( ) ±56.7 ( ) ±66.1 ( ) ±1. (1. -.0) ±1.6 ( ) ±1.5 (1.5 -.) ±0.7 ( ) ±38.6 ( ) ±7.7 ( ) ±157 ( ) 1. 3.±. ( ) ±.3 ( ) ±3.9 (.3 -.8) ±56.3 ( ) ±36.1 ( ) ±3.7 ( ) ±73.7 ( ) ±16.0 ( ) ±.7 ( ) ±5.1 (3.6-7.) ±7.9 ( ) ±9.6 (.0-90.) ±57. ( ) ±39.7 ( ) ±100 ( ) ±35.6 ( ). 3.5±.5 (.3 -.7) ±1. ( ) Supplementary Table.