Detec%ng  Unique  Proteins  in  Rheumatoid  Arthri%s    Pa%ents  with  Inters%%al  Lung  Disease  

Introduction Rheumatoid   Arthritis   is   a   systemic   autoimmune   disease  which   primarily   causes   in5lammation   in  joint   tissues.   Other   tissues   of   the   body   are   affected   by   this   disease   as  well   though,   such   as   lung  tissue,   which   was   the   focus   of   this   study.   In5lammation   in   the   lungs   caused   by   RA   can   lead   to  Interstitial  Lung  Disease  (ILD),  a  broad  array  of  complications  which  affect  10-­‐20%  of  RA  patients  and  can  be  fatal.  Currently,  the  cause  of  the  autoimmune  response  in  the  lung  tissues  is  unknown,  but   it   has   been   hypothesized   that   citrullinated   proteins   (proteins   that   have   had   the   amino   acid  arginine   converted   to   citrulline   by   the   enzyme   peptidylarginine   deiminase)   could   represent   the  basis  of  this  autoimmune  response.  While  the  underlying  origin  of  protein  citrullination  in  the  lungs  is  not   fully  understood  either,   these  proteins  nevertheless  represent  a  potentially   inexpensive  and  noninvasive  biomarker  that  could  be  used  in  early  detection  of  RA-­‐ILD.    

Question Do   RA   patient   with   ILD   possess   unique   citrullinated   protein   pro5iles  compared  to  patients  without  ILD?  

Alexander Davis Hurley1, Phu Van1, Dana Ascherman, M.D.2, 3, John Minden1 1Department of Biological Sciences, Carnegie Mellon University

2Division of Rheumatology, University of Miami 3Miami VA Medical Center

Figure 1. An image of a chest x-ray of a patient with idiopathic interstitial pneumonia – a disease that is considered a part of the Interstitial Lung Diesease category.

http://casereports.bmj.com/content/2010/bcr.02.2010.2765/F1.large.jpg

Materials Blood  serum  samples  were  acquired  from  the  two  subgroups  of  patients  (RA-­‐No  ILD  and  RA-­‐ILD).  The  RA-­‐No   ILD  category   included   samples  15  and  16,   and   the  RA-­‐ILD  category   included   samples  HAW,  27,  and  28.  While  these  serum  samples  did  not  contain  high  levels  of  the  proteins  in  question,  they  did  contain  the  antibodies  produced  by  the  patient’s  immune  system  to  target  the  citrullinated  proteins.  Because  they  have  high  binding  speci5icity,  and  because  they  are  more  abundant  than  the  citrullinated   proteins,   these   antibodies   were   considered   to   be   viable   proxies   for   patient-­‐speci5ic  protein   samples.   The   citrullinated   and   non-­‐citrullinated   proteins   used   in   the   experiment   were  instead  produced  though  in  vitro,  PAD-­‐catalyzed  deimination  of  cell  lysates  prepared  from  K562  and  various  lung-­‐derived  cell  lines  (Ascherman).    

Methods 1)  Binding:  • Mix   the   sample   of   one   patient   with   Protein   A   sepharose   beads   to   allow   the  antibodies  in  the  sample  to  bind  with  the  beads  • Split  antibody-­‐bead  complex  into  two  components  • Bind   one   component   with   citrullinated   protein   extract   and   the   other   with  uncitrullinated  protein  extract  

2)  Two  Dimensional  Difference  Gel  Electrophoresis:  • Minimally   label   citrullinated   samples   with   one   5luorescent   cyanide   dye   and  uncitrullinated  samples  with  another  (Cy3  and  Cy5)  • Combine  the  two  samples  together  • Isoelectrically  focus  the  mixture  on  a  pH  3-­‐10  gel  strip  • Co-­‐electrophorese  the  pH  separated  proteins  on  a  polyacrylamide  gel  

3)  Imaging:  • Fluorescently  image  each  dye  channel  of  the  gel  (Cy3  and  Cy5)    • Combine   these   pictures   into   an   overlay   (see   overlay   example   below)   that   allows  difference  proteins  to  be  identi5ied  among  background  protein  • Compare  this  overlay  with  the  overlay  from  other  patients  and  determine  unique  differences  between  categories      

Results

Figure  2.   This   image  depicts   the   citrullination  of   a   protein.   The   change   in  protein   structure   occurs  when   the  oxygen  atom  replaces  the  nitrogen  and  the  hydrogen  located  at  the  top  right  of  the  structure  depicted  here.  

http://upload.wikimedia.org/wikipedia/commons/thumb/b/b6/Citrullination.svg/400px-­‐Citrullination.svg.png  

Blood Serum Sample

Mix with Citrullinated

Protein Extracts

Mix with Uncitrullinated

Protein Extracts

Figure  3.  This  5low  chart  outlines  the  binding  and  2D-­‐DIGE  steps  of  the  experimental  protocol.  This  diagram  was  inspired  by  a  similar  5igure  in  Gong,  et.  al.’s  Drosophila  Ventral  Furrow  Morphogenesis:  A  Proteomic  Analysis.  2004.    

Conclusions and Future Work This  project  represents  one  of  the  5irst  attempts  to  analyze  the  blood  serum  of  these  Rheumatoid  Arthritis  patients  with  the  2D-­‐DIGE  technique.  Because  of  this,  it  is  dif5icult  to  conclude  with  certainty  that  the  differences  detected  between  the  RA-­‐ILD  and  RA-­‐No  ILD  categories  represent  reliable  indicators  of  the  disease  complication.  Looking  at  the  data  gathered  though,  one  must  conclude  that  the  ILD  category  did  not  have  unique  citrullinated  spots  that  distinguished  these  patient  from  those  with  RA-­‐No  ILD.  However,  it  did  appear  that  the  RA-­‐No  ILD  category  possessed  more  unique  uncitrullinated  or  mixed  spots  than  the  other  category.  Perhaps  the  absence  of  these  spots  in  the  RA-­‐ILD  category  could  represent  an  important  indicator  of  the  complication.  To  know  for  sure,  several  aspects  of  the  experimental  design  could  be  altered  or  otherwise  improved  upon   in  order   to  more  effectively  answer   the   initial  question  of   the  project.  First,   removing   the  antibodies   from  the   5inal  protein  mixture  prior  to  isoelectric  focusing  and  electrophoresis  would  signi5icantly  clear  up  background  5luorescence  in  the  gel   images.   With   less   background   clutter   in   the   image   it   is   possible   that   more   difference   proteins   could   be   identi5ied  between   the   RA-­‐ILD   and   RA-­‐No   ILD   samples   used   in   this   experiment.   Another   alteration   to   the   experimental   design   of  particular  interest  is  the  co-­‐electrophoresing  of  samples  from  different  patients.  For  example,  sample  16  and  sample  HAW  (No-­‐ILD   and   ILD   categories,   respectively)   could   both   be   combined   with   citrullinated   proteins,   labeled,   and   then   co-­‐electrophoresed.  This  method  would  allow  for  an  even  more  direct  comparison  of   the  citrullinated  protein  pro5iles  of   the  patients  involved  in  the  study.    

Acknowledgements I  am  indebted  to  the  Minden  lab,  the  NSF-­‐REU  coordinators  at  Carnegie  Mellon,  the  National  Science  Foundation,  Centre  College,  and  the  James  G.  Brown  Foundation  for  all  the  support  this  summer.  

References Ascherman,  D.,  et.  al.,  Peripheral  Blood  Biomarkers  of  Rheumatoid  Arthritis-­‐Associated  Interstitial  Lung  Disease.  2010.  

No-­‐ILD   ILD  

Sample  16   Sample  HAW   Sample  27   Sample  28  

1  

2  

3  

Citrullinated  Protein:  Green          Uncitrullinated  Protein:  Red            Mix  of  Cit./Uncit.  Protein:  Yellow  

No-­‐ILD   ILD  

Sample  16     Sample  HAW   Sample  27   Sample  28    

1  

2  

3  

Discussion • Note:   Sample   15,   which   was   in   the   No-­‐ILD   category,   was   removed   from   5inal   analysis   because   the   gel   was   poorly  isoelectrically  focused  and  could  not  be  accurately  compared  with  other  gels.  

Table  1  displays  three  protein  groups  that  are  common  to  both  categories  of  RA  patient.  • Group  number  1  shows  two  citrullinated  protein  spots  that  were  common  to  the  upper  left  hand  corner  of  the  gels.    • Group  number  2  shows  a  string  of  citrullinated  and  uncitrullinated  proteins  in  the  upper  middle  of  the  gels.    • Group  number  3  shows  a  string  of  citrullinated  proteins  common  to  all  gels  in  the  middle  region  of  all  the  gels.  

Table  2  displays  three  protein  groups  that  are  unique  to  one  gel  or  another.    • Group  1  of  this  table  shows  an  uncitrullinated  protein  spot  in  sample  16  that  is  not  present  in  the  ILD  category  at  all.    • Group  2  shows  that  sample  16  and  HAW,  although  from  patients  in  different  categories,  have  several  prominent  citrullinated  spots  in  the  lower  portion  of  the  gel  that  are  unique  from  samples  27  and  28.    • Group   3   shows   a   very   bright   set   of  mixed   (Citrullinated   and   Uncitrullinated)   proteins   in   samples   16.   The   ILD   gels  may  display  this  same  set  of  proteins,  but  they  are  very  faint  when  comparing  images  that  are  matched  for  intensity.    

Figure 4. An example of a full gel overlay. Pictured here is sample 16.

Table  1.  Three  Similar  Protein  Spots  Among  Both  Categories  of  Patient   Table  2.  Three  Unique  Protein  Spots  

Citrullinated  Protein:  Green  Uncitrullinated  Protein:  Red  

Mix  of  Cit./Uncit.  Protein:  Yellow