K V F G R C E L A A A M K R H G L D N Y R G Y S L G N W V C A A K F E S N F N T Q A T N R N T D G S T D Y G I L Q I N S R W W C N D G R T P G S R N L C N I P C S A L L S S D I T A S V N C A K K I V S D G N G M N A W V A W R N R C K G T D V Q A W I R G C R LK V F G R C Y R G Y S L G N G S T D Y G I L Q I N S R G R T P G S R N L C N I P C S A N G M N A W V A W R N R C KK V F G R C E L G Y S L G N W V C A A K F E S N D Y G I L Q I N S G R T P G S R N L C N I P C S A L G M N A W V A W R N R C KK V F G R C E L A S L G N W V C A A K F E Y G I L Q I N S R W W C T P G S R N L C N I N A W V A W R N

G R C E L A A A M K R H L G N W V C A A K F E S N Y G I L Q I N S R W W C N S R N L C N I P C S A L L S S D I A W V A W R N R C K G T D V QG R C E L A A A M K R H G L G N W V C A A K F E S N L Q I N S R W W C N S R N L C N I P C S A L L S S D I T A S V W V A W R N R C K G T D

R C E L A A A M K R H G C A A K F E S N F N T Q A T L Q I N S R W W C N D G R T P G N I P C S A L L S S D I T A S V W V A W R N R C K G T D V QC E L A A A M K R H A K F E S N F N Q I N S R W I P C S A L L S S D I T A S V W V A W R N R C K G T D V Q A

E L A A A M K R H G F E S N F N T Q A Q I N S R W W C N D G R T S S D I T A S V N C A K K I V S D G V A W R N R C K G T DL A A A M K R H G L D N Y R E S N F N T Q A T N R N T Q I N S R W W C N D G R T P G S S D I T A S V N C A K K I V S D G N G M N A W V A W R

A A A M K R H G L D N Y R G Y N F N T Q A T N R Q I N S R W W C N D G R T P G S R N L C N S D I T A S V N C A K K I V S D G V A W R N R C K G T D V QA A M K R H G L D N Y R G N F N T Q A T N R N T D G S T W C N D G R T P G S R N A S V N C A K K I V S D G N G M N A V A W R N R C K G T D V Q A

A M K R H G L D N Y F N T Q A T N R N T D G S T W C N D G R T P G S R N L S V N C A K K I V S D G A W R N R C K G T D V Q AA M K R H G L D N Y R G N T Q A T N R N T D G S T D W C N D G R T P G S R N L C N S V N C A K K I V S D G N G M W R N R C K G T D V Q AA M K R H G L D N Y R G Y S L G N N T Q A T N R N T D G S T D Y W C N D G R T P G S R N L C N I P C S A S V N C A K K I V S D G N G M N A W I R G C R L

M K R H G L D N Y R G N T Q A T N R N T D G S T D Y G I W C N D G R T P G S R N L C N I P C S A L S V N C A K K I V S D G N G M N A W I R G C R LM K R H G L D N Y R G Y S N T Q A T N R N T D G S T D Y G I L W C N D G R T P G S R N L C N I P C S A L L S V N C A K K I V S D G N G M N A W

L D N Y R G Y S L G N W V C R N T D G S T D Y G I W C N D G R T P G S R N L C N I P C S A L L S S D I T A C A K K I V S D G N G M N AL D N Y R G Y S L G N W V C A R N T D G S T D Y G I L Q I N S R C N D G R T P G S R N L C N I A K K I V S D G N G M N A

D N Y R G Y S L G N W V C A A K F E S N T D G S T D Y G I L C N D G R T P G S R N L C N I P C S A L L S S D I T A S V A K K I V S D G N G M N A WC N D G R T P G S R N L C N I P C S A L L S S D I T A S V N K K I V S D G N G M N A W V A

N D G R T P G S R N L C N I P C S A L L S S D I T A S V N C K I V S D G N G MD G R T P G S R N L C N I P C S

G R T P G S R N L C N I P

K V F G R C E L A A A M K R H G L D N Y R G Y S L G N W V C A A K F E S N F N T Q A T N R N T D G S T D Y G I L Q I N S R W W C N D G R T P G S R N L C N I P C S A L L S S D I T A S V N C A K K I V S D G N G M N A W V A W R N R C K G T D V Q A W I R G C R LK V F G R C E L G Y S L G N W V C A A K F R N T D G S T D Y G I L Q I S S D I T A N A W V A W R N R CK V F G R C E L A A A M S L G N W V C A A K F S T D Y G I L Q I N S R W D I T A S V N C A K K I V V A W R N R C K G T D V Q A W

G R C E L W V C A A K F G I L Q I N S R W W A S V N C A K K I V S D G N G M A W R N R C K G T D V Q A WR C E L A A A M K R H G L A A K F E S N F N T Q W W C N D G R S V N C A K K I V S D G N G M R N R C K G T D V Q A W

L A A A M K R H G L D A A K F E S N F N T Q A T N W W C N D G R T P G S R C A K K I V S D G N G M N A N R C K G T D V Q A WL A A A M K R H G L D N Y R A K F E S N F N T Q C N D G R T P G S R C A K K I V S D G N G M N A W R C K G T D V Q A

A A A M K R H G L D A K F E S N F N T Q A N D G R T P G S R N L C K I V S D G N G M C K G T D V Q AK R H G L D N Y R G Y K F E S N F N T Q A T N R N G S R N L C N I P C S A L K I V S D G N G M N A C K G T D V Q A W

H G L D N Y R G Y E S N F N T Q A T N R N T D G S T D S R N L C N I P K I V S D G N G M N A W G T D V Q A W I R GG L D N Y R G Y S L G N N F N T Q A T N R N T D G S N L C N I P C S A L K I V S D G N G M N A W V A W W I R G C R L

Y R G Y S L G N W V C R N T D G S T D Y G I N I P C S A L L S S D I K I V S D G N G M N A W V A W R N I R G C R LI V S D G N G M N AI V S D G N G M N A WI V S D G N G M N A W V A W

100 110 12050 60 70 80 901 10 20 30 40

1 10 20 30 40 90 100 110 12050 807060

CIAL

Miralles. B., Benedé. S., Jiménez-Saiz, R., López-Expósito, I., Molina, E., López-Fandiño, R.Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), Madrid, Spain

Lysozyme (LYS) is a major allergen of egg white, although its allergenic potential has not been studied in depth and no relevant epitopes have been identified as yet. This investigation addresses the identification of the peptides produced upon gastrointestinal digestion of LYS and their IgE-binding, as a contribution to the understanding of what makes LYS a potent allergen.

To projects COST-Infogest FA1005 and AGL2011-24740. S.B. and I. L-E acknowledge the financial support of CSIC through JAE Pre and JAE Doc grants.

The peptide profiles suggest that, in addition to the 1.95, 3.06 and 1.78 KDa immunoreactive fragments, gastric digestion could generate peptides linked by disulfide bonds retaining reactive IgE epitopes. The fragment LYS (24-129) remains unhydrolyzed during duodenal digestion, being a good candidate to be absorbed and interact with the immune system. The behaviour of LYS during gastroduodenal digestion and, in particular, its dependence on gastric pH likely influences the sensitizing and eliciting capacity of the protein

IDENTIFICATION OF IgE REACTIVE FRAGMENTS IN

HEN EGG LYSOZYME

Pepsin hydrolysed LYS more slowly at pH 2.0 than at pH 1.5 and there was intact LYS surviving pepsin action after 60 min at pH 2.0 but not at pH 1.5. Upon subsequent duodenal digestion, the 4 KDa band was fully degraded, whereas, intact LYS that resisted gastric digestion at pH 2.0, partially precipitated during the duodenal stage at pH 6.5 and a new 12 kDa band, that exhibited a considerably IgE binding, was formed.

RESULTS: PROTEOLYSISOBJECTIVE

MATERIAL AND METHODS

RESULTS: IMMUNOREACTIVITY

RESULTS: PEPTIDE AND EPITOPE IDENTIFICATION

ACKNOWLEDGMENTS

Figure 2. SDS-PAGE under reducing conditions and Immunoblotting of LYS (1) subjected to gastric digestion for 60 min at pH 1.5 (2) and 2.0 (3).

Figure 1. RP-HPLC patterns of LYS subjected to gastric digestion at pH 1.5 and 2.0.

Gastric 30 min

pH 1.5 pH 2.0

Gastric 30 min

Gastric 60 min Gastric 60 min

Figure 3. Immunoblotting of LYS (1) and its duodenal digests following gastric digestions at pH 1.5 (2) and 2.0 (3) with ten individual sera.

The digestion was performed in two steps by using simulated gastric and duodenal fluids. The analysis of the samples was performed by RP-HPLC, SDS-PAGE, Immunoblotting with sera from egg-allergic patients and RP-HPLC-MS/MS. For intact molecular weight determination of reactive bands proteins were electroblotted onto a nitrocellulose membrane and directly analyzed by MALDI-TOF. The identification of the peptides was performed by MALDI-TOF/TOF.

CONCLUSIONS

The peptides identified by RP-HPLC-MS/MS in the gastric digests of LYS at pH 2.0 for 60 min and subsequent duodenal digest for 30 min are shown below, together with the sequences identified by MALDI-TOF/TOF after in-gel tryptic digestion of the LYS fragments recognized by human IgE derived

from gastric and gastroduodenal digestion, highlighted in the primary sequence of the protein.

Undetected

Immunoblotting showed the

appearance of an IgE-reactive band

of approximately 4 kDa after gastric digestions at pH

1.5 and 2.0.