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Naturally processed C-MYC protein-derived peptide bound by HLA class I molecules
Abstracts
3
B-3.1 #54
NATURALLY P R O C E S S E D C-MYC PROTEIN-DERIVED PEPTIDE BOUND BY HLA CLASS I MOLECULES. p, Harris, A. Colovai, Z. Liu, R. Dalla F a v e r a a n d N. S u c i u - F o c a . D e p a r t m e n t of Pathology, C o l u m b i a University, New York, NY T h e h y p o t h e s i s t h a t t r a n s f o r m a t i o n r e s u l t s in a c h a n g e in t h e profile of p e p t i d e s b o u n d to MHC c l a s s I m o l e c u l e s w a s t e s t e d u s i n g a l y m p h o b l a s t o i d B cell llne (HLA-A2, A68, B40) t r a n s f e c t e d with a transcriptionally-activated form of c-myc. Naturallyp r o c e s s e d p e p t i d e s , b o u n d to HLA c l a s s I m o l e c u l e s , w e r e isolated, f r o m b o t h t h e c - m y c t r a n s f e c t e d LBCL a n d t h e v e c t o r a l o n e t r a n s f e c t e d c o u n t e r p a r t , for s e q u e n c e a n a l y s i s . F o r t y - t h r e e sequences of bound peptides could be grouped into three structural motifs. One of the peptide sequences obtained, SLLPAIVEV, w a s i d e n t i c a l to a p r e v i o u s l y r e p o r t e d p e p t i d e b o u n d to HLA-A2.1. a n d w a s u s e d a s a t e m p l a t e for g r o u p i n g HLA-A2 bound peptides. A s e c o n d m o t i f , i d e n t i c a l to t h a t p r e v i o u s l y r e p o r t e d for H L A - A 6 8 - b o u n d p e p t i d e s , w a s a l s o o b s e r v e d . A d i s t i n c t t h i r d motif, c o n s i s t e n t w i t h t h e s t r u c t u r e of t h e HLA-B40 "45 p o c k e t " , w a s o b s e r v e d . T h e p e p t i d e s w i t h i n t h i s g r o u p c o n t a i n e d g l u t a m a t e in p o s i t i o n 2, u s u a l l y f o l l o w e d b y a h y d r o p h o b i c r e s i d u e in p o s i t i o n s t h r e e a n d n i n e . W i t h i n t h i s g r o u p of p e p t i d e s b o u n d to MHC C l a s s I m o l e c u l e s , o n e peptide, HEETPPTTS, w a s 100o/6 h o m o l o g o u s to r e s i d u e s 2 4 1 - 2 4 9 of t h e c-myc p r o t e i n a n d u n i q u e to t h e c-myc t r a n s f e c t e d LBCL. H e n c e , oncogene products are processed and presented by MHC-class I molecuels.
B-3.1 #60
MHC CLASS II MOLECULES EXPRESS A TARGETING SIGNAL THAT DIRECTS LOCALIZATION TO THE ENDOCYTIC COMPARTMENTS OF ANTIGEN PRESENTING CELLS. Andrea 1. Sant and Alexander Chervonsky. University of Chicago, Chicago, IL Although it is generally accepted that the ability of Class 11molecules to present exogenous antigen reflects its selective ability to traffic to endocytic compartments of APC, the structural features in the Class II that direct it into these compatm~ents are a subject of debate and include at least two possibilities: sequences in the Class II molecule itself or sequences in the associated invariant giycoprotein (Ii). The finding that cells lacking li are capable of presenting exogenous antigens suggested to us that Class 11molecules access the endosornal compartments independently of Ii. We have used site directed mutagenesis, transfection, and intracellular staining to identify sequences in the class II molecule that control this localization. We have found that, independently of Ii, Class II localization within the endocytic compartments can be either positively or negatively modulated by single, conservative amino acid changes in a higiy conserved region of the 13chain. Compared to WT class II, which is expressed primarily in the RER and Golgi with limited expression in late endosornes, mutation at position 82 causes intracellular arrest in. late golgi compartments, mutation at position 81 causes a loss in expression within the endocytic pathway but no loss in cell surface expression, while mutation at position 80 leads to dramatic enrichment in the endocytic compartments which co-stain with the late endosomal marker LAMP- 1. Cotransfeetion of Ii into cells expressing the mutant class II shifts 81m to a totally vesicular pattern, while 82m remains in the golgi, indicating that Ii only functions in the context of the appropriate Class II sequence. Collectively our data suggests a model of Class II transport in which the 80-84 sequence of 13controls sorting into the endocytic pathway, with Ii providing an endosomal retention, rather than a dominant targeting signal.
3
B-3.1 #54
NATURALLY P R O C E S S E D C-MYC PROTEIN-DERIVED PEPTIDE BOUND BY HLA CLASS I MOLECULES. p, Harris, A. Colovai, Z. Liu, R. Dalla F a v e r a a n d N. S u c i u - F o c a . D e p a r t m e n t of Pathology, C o l u m b i a University, New York, NY T h e h y p o t h e s i s t h a t t r a n s f o r m a t i o n r e s u l t s in a c h a n g e in t h e profile of p e p t i d e s b o u n d to MHC c l a s s I m o l e c u l e s w a s t e s t e d u s i n g a l y m p h o b l a s t o i d B cell llne (HLA-A2, A68, B40) t r a n s f e c t e d with a transcriptionally-activated form of c-myc. Naturallyp r o c e s s e d p e p t i d e s , b o u n d to HLA c l a s s I m o l e c u l e s , w e r e isolated, f r o m b o t h t h e c - m y c t r a n s f e c t e d LBCL a n d t h e v e c t o r a l o n e t r a n s f e c t e d c o u n t e r p a r t , for s e q u e n c e a n a l y s i s . F o r t y - t h r e e sequences of bound peptides could be grouped into three structural motifs. One of the peptide sequences obtained, SLLPAIVEV, w a s i d e n t i c a l to a p r e v i o u s l y r e p o r t e d p e p t i d e b o u n d to HLA-A2.1. a n d w a s u s e d a s a t e m p l a t e for g r o u p i n g HLA-A2 bound peptides. A s e c o n d m o t i f , i d e n t i c a l to t h a t p r e v i o u s l y r e p o r t e d for H L A - A 6 8 - b o u n d p e p t i d e s , w a s a l s o o b s e r v e d . A d i s t i n c t t h i r d motif, c o n s i s t e n t w i t h t h e s t r u c t u r e of t h e HLA-B40 "45 p o c k e t " , w a s o b s e r v e d . T h e p e p t i d e s w i t h i n t h i s g r o u p c o n t a i n e d g l u t a m a t e in p o s i t i o n 2, u s u a l l y f o l l o w e d b y a h y d r o p h o b i c r e s i d u e in p o s i t i o n s t h r e e a n d n i n e . W i t h i n t h i s g r o u p of p e p t i d e s b o u n d to MHC C l a s s I m o l e c u l e s , o n e peptide, HEETPPTTS, w a s 100o/6 h o m o l o g o u s to r e s i d u e s 2 4 1 - 2 4 9 of t h e c-myc p r o t e i n a n d u n i q u e to t h e c-myc t r a n s f e c t e d LBCL. H e n c e , oncogene products are processed and presented by MHC-class I molecuels.
B-3.1 #60
MHC CLASS II MOLECULES EXPRESS A TARGETING SIGNAL THAT DIRECTS LOCALIZATION TO THE ENDOCYTIC COMPARTMENTS OF ANTIGEN PRESENTING CELLS. Andrea 1. Sant and Alexander Chervonsky. University of Chicago, Chicago, IL Although it is generally accepted that the ability of Class 11molecules to present exogenous antigen reflects its selective ability to traffic to endocytic compartments of APC, the structural features in the Class II that direct it into these compatm~ents are a subject of debate and include at least two possibilities: sequences in the Class II molecule itself or sequences in the associated invariant giycoprotein (Ii). The finding that cells lacking li are capable of presenting exogenous antigens suggested to us that Class 11molecules access the endosornal compartments independently of Ii. We have used site directed mutagenesis, transfection, and intracellular staining to identify sequences in the class II molecule that control this localization. We have found that, independently of Ii, Class II localization within the endocytic compartments can be either positively or negatively modulated by single, conservative amino acid changes in a higiy conserved region of the 13chain. Compared to WT class II, which is expressed primarily in the RER and Golgi with limited expression in late endosornes, mutation at position 82 causes intracellular arrest in. late golgi compartments, mutation at position 81 causes a loss in expression within the endocytic pathway but no loss in cell surface expression, while mutation at position 80 leads to dramatic enrichment in the endocytic compartments which co-stain with the late endosomal marker LAMP- 1. Cotransfeetion of Ii into cells expressing the mutant class II shifts 81m to a totally vesicular pattern, while 82m remains in the golgi, indicating that Ii only functions in the context of the appropriate Class II sequence. Collectively our data suggests a model of Class II transport in which the 80-84 sequence of 13controls sorting into the endocytic pathway, with Ii providing an endosomal retention, rather than a dominant targeting signal.