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Results

   Aim One:  

  M3 antigenic peptides
There was a total of 578 possible 13mer peptides from the 590 amino acid muscarinic receptor (shown on the right).

Each letter codes for an amino acid.
MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTV
THFGSYNVSRAAGNFSSPDGTTDDPLGGHTVW
QVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVN
NYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNL
ACDLWLAIDYVASNASVMNLLVISFDRYFSITRP TYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQ
YFVGKRTVPPGECFIQFLSEPTITFGTAIAAFYMPV
TIMTILYWRIYKETEKRTKELAGLQASGTEAETENF
VHPTGSSRSCSSYELQQQSMKRSNRRKYGRCHF
WFTTKSWKPSSEQMDQDHSSSDSWNNNDAAAS
LENSASSDEEDIGSETRAIYSIVLKLPGHSTILNSTK
LPSSDNLQVPEEELGMVDLERKADKLQAQKSVDD
GGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKS
TATLPLSFKEATLAKRFALKTRSQITKRKRMSLVK
EKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIP
KTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFK
MLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL

Click to enlarge.
Figure 4. The calculated energy values, indicating stability of the peptide bound I-Ag7 structures. The lower energy values indicate a higher affinity between the antigenic peptide and the MHC class II (I-Ag7) molecule.

  First Elimination
From these energy values, peptides with values falling below -145 Joules were chosen as likely candidates capable of binding with the MHC Class II molecule in vivo.
  View Data
  HTML file (Table 1)

  Key
  Green: (-145 to -150 J)
  Yellow: (-150 J and below)

Note: Between -145 and -150 J, only antigenic peptides likely to quality for Second Elimination were chosen.

Figure 5. Example of a low energy (likely) antigenic peptide. 1ES0_254 Notice that the peptide nicely follows the curvature of the binding groove.

Figure 6. Example of a high energy (unlikely) antigenic peptide. 1ES0_243 Notice that the peptide's bulky aromatic groups do not allow the peptide to fit nicely into the binding groove.


  Second Elimination
The Second Elimination was based on the location of the actual peptide in relation to the cell's membrane. The peptide must be found in the extracell-ular environment in order to be capable of activating an immune response, either through (1) endocytosis or (2) uptake by antigen presenting cells (APCs).
  View Data
The location of the actual peptide was determined from:

  (a) Hydropathy Profile*1

  (b) M3 Homology Model*2
       (Figure 7)

  Sources
1 - SOSUI database
2 - D Escuela, Young Bioinformaticians Forum

Figure 7. M3 muscarinic receptor homology model. The likely candidates (facing the extracellular side) are peptides 15, 131, and 507 from the M3 muscarinic receptor. Peptide 94, 254, and 523 are good examples of peptides that would qualify through first eliminations due to their stability , but because of their physical locations are unlikely to be antigenic peptides.
Click to enlarge.
 
Likely Candidates
Peptide 15:   NISSSWIHSPSDA
Peptide 131: NRWALGNLACLW
Peptide 507: YNIMVLVNTFCOS

Note:
The MHC class II presentation pathway makes it probable that only peptides in the extracellular part of a transmembrane protein are likely to be processed for binding to the MHC molecule.



   Aim Two:  

Figure 8. Comparison of the M3 receptor (green) found within the lacrimal cells mice indicated. The red marker indicates rhodamine phalloidin staining, which stains actin filaments.
 
(a) BALB/C normal mice. 1 month old.
 
(b) BALB/C normal mice. 4 months old.
 
(c) NOD diseased mice. 1 month old.
 
(d) NOD diseased mice. 4 months old.

   Lysosomal Staining
The following video is a 3D look at the lysosomal cavities (red) within the lacrimal cells. The green marker indicates FITC-phalloidin staining, which stains actin filaments.

Video Link (Macromedia Flash Format):

   1 month NOD diseased mice

   4 month NOD diseased mice

   1 month BALB/C normal mice

   4 month BALB/C normal mice

With great difficulty, the lysosomal markers were re-stained to show the following videos below. The lysosomal cavities are stained red, and the the magenta marker indicates phalloidin staining.

   1 month BALB/C normal mice

   4 month BALB/C normal mice

    Result Summary
There was great difficulty in co-staining the Rab7 antibody (anti-lysosomal cavity/ rabbit host) with the M3 antibody (anti-muscarinic receptor/ goat host). In essence our major concern is that both our best muscarinic antibody and our best lysosomal antibody are rabbit host. This makes it impossible to co-stain with our best antibodies. As a result, we have chosen a second-class goat host antibody for the muscarinic receptor which hasn't shown very much success when immunostaining frozen sections. However, after careful analysis of the staining with lysosomal cavities, it is still very much in my opinion that there is a great possibility for support of our hypothesis - I still firmly believe that the lysosomal cavities can show co-localization with the muscarinic receptor. Nonetheless, this theory must still be supported by physical co-localization in order to be seriously concluded.

Glossary

   13mer
   A
   Antigen
   Autoantigen
   B
   BALB/C mice
   C
   Confocal Microscopy
   G
   GAD
   H
   Hydropathy Profile
   I
   I-Ag7
   Immunostaining
   M
   M3 receptor
   MHC Class II
   N
   NOD mice
   S
   Sjögren's Syndrome
 

 

 A Arianjam, A Schiewe, I Haworth, & SF Hamm-Alvarez
USC School of Pharmacy
 © 2005

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