Complement

 system  

Santosh  Khanal  
 Jules  Bordet,  Pasteur  Ins8tute  
•  Sheep   an8serum   to   the   bacterium   Vibrio  
cholerae  caused  lysis  of  the  bacteria  
•  Hea8ng   the   an8serum   destroyed   its  
bacterioly8c  ac8vity  
•  The  ability  to  lyse  the  bacteria  was  restored  to  
the  heated  serum  by  adding  fresh  serum  that  
contained   no   an8bodies   directed   against   the  
bacterium   and   was   unable   to   kill   the  
bacterium  by  itself  

Santosh  Khanal  
 Jules  Bordet,  Pasteur  Ins8tute  
•  Bacterioly8c   ac8vity   requires   two   different  
substances:    
Ø the  specific  an8bacterial  an8bodies,  which  survive  
the  hea8ng  process  
Ø a   heat-­‐sensi8ve   component   responsible   for   the  
ly8c  ac8vity  

Santosh  Khanal  
 Paul  Ehrlich,  Berlin    
•  Independently   carried   out   similar   experiments  
and  coined  the  term  complement  
•  Defined  it  as  “the  ac8vity  of  blood  serum  that  
completes  the  ac8on  of  an8body”    

Santosh  Khanal  
 Complement  
•  Refers   to   a   system   of   factors,   which   occur   in  
normal  serum  
•  More  than  30  soluble  and  cell-­‐bound  proteins    
•  Ac8vated   characteris8cally   by   Ag-­‐Ab  
interac8on  
•  Mediate   a   number   of   biologically   significant  
consequences  

Santosh  Khanal  
 Func8ons  of  complement  
•  Lysis  of  cells,  bacteria,  and  viruses  
•  Opsoniza8on,   which   promotes   phagocytosis   of  
par8culate  an8gens  
•  Binding  to  specific  complement  receptors  on  cells  
of   the   immune   system,   triggering   specific   cell  
func8ons,   inflamma8on,   and   secre8on   of  
immunoregulatory  molecules  
•  Immune   clearance,   which   removes   immune  
complexes   from   the   circula8on   and   deposits  
them  in  the  spleen  and  liver  
Santosh  Khanal  
Mul8ple  ac8vi8es  of  the  complement  
system  

Santosh  Khanal  
 The  Complement  Components  
•  The  proteins  and  glycoproteins  
•  Synthesized  by:  
Ø liver  hepatocytes  
Ø blood  monocytes  
Ø 8ssue  macrophages  
Ø e pithelial   cells   of   the   gastrointes8nal   and  
genitourinary  tracts  
•  Cons8tute  5%  of  the  serum  globulin  frac8on  

Santosh  Khanal  
 The  Complement  Components  
•  Most   circulate   in   the   serum   in   func8onally  
inac8ve  forms  as  proenzymes  or  zymogens  
•  Inac8ve   un8l   proteoly8c   cleavage,   which  
removes   an   inhibitory   fragment   and   exposes  
the  ac8ve  site  
•  The   complement-­‐reac8on   sequence   starts  
with  an  enzyme  cascade  

Santosh  Khanal  
 The  Complement  Components  
•  Designated   by   numerals   (C1–C9)   or   by   le^er  
symbols  (factor  D)  
•  Pep8de   fragments   formed   by   ac8va8on   of   a  
component  are  denoted  by  small  le^ers  
•  In   most   cases,   the   smaller   fragment   is  
designated   “a”   and   the   larger   fragment  
designated   “b”   (C3a,   C3b;   except   C2:   C2a   is  
the  larger  cleavage  fragment)  

Santosh  Khanal  
 The  Complement  Components  
•  The  larger  fragments  bind  to  the  target  near  the  
site  of  ac8va8on  
•  The   smaller   fragments   diffuse   from   the   site   and  
can  ini8ate  localized  inflammatory  responses  
•  The   complement   fragments   interact   with   one  
another  to  form  func8onal  complexes  
•  Complexes   having   enzyma8c   ac8vity   are  
designated   by   a   bar   over   the   number   or   symbol  
(C4b2a,  C3bBb)  
Santosh  Khanal  
 Complement  Ac8va8on  
1.  Classical  pathway  
2.  Alterna8ve  pathway  
3.  Lec8n  pathway  

Santosh  Khanal  
Complement  ac8va8on  pathways  

Santosh  Khanal  
 Classical  pathway    
•  Begins   with   forma8on   of   soluble   an8gen-­‐
an8body  complexes  (immune  complexes)  
•  Binding   of   an8body   to   an8gen   on   a   suitable  
target,  such  as  a  bacterial  cell  
•  Involves   IgM   and   certain   subclasses   of   IgG  
(human  IgG1,  IgG2,  and  IgG3)  

Santosh  Khanal  
 Classical  pathway  
•  The   ini8al   stage   of   ac8va8on   involves   C1,   C2,  
C3,  and  C4  
•  Present   in   plasma   in   func8onally   inac8ve  
forms  
•  The  forma8on  of  an  an8gen-­‐an8body  complex  
induces   conforma8onal   changes   in   the   Fc  
por8on  of  the  IgM  molecule    
•  Expose  a  binding  site  for  the  C1  component  of  
the  complement  system  

Santosh  Khanal  
 C1qr2s2  
•  C1  in  serum  is  a  macromolecular  complex    
•  Consists  of  C1q  and  two  molecules  each  of  C1r  
and  C1s,  held  together  in  a  complex  (C1qr2s2)  
stabilized  by  Ca2+  ions  
•  The   C1q   molecule   is   composed   of   18  
polypep8de  chains  

Santosh  Khanal  
C1qr2s2  contd.  

•  The   chains   associate   to   form   six   collagen-­‐like  

triple  helical  arms  
•  The   8ps   bind   to   exposed   C1q-­‐binding   sites   in  
the  CH2  domain  of  the  an8body  molecule  
•  Each   C1r   and   C1s   monomer   contains   a  
cataly8c  domain  and  an  interac8on  domain    
•  The   interac8on   domain   facilitates   interac8on  
with  C1q  or  with  each  other  

Santosh  Khanal  
Structure  of  the  C1  macromolecular  
complex  

Santosh  Khanal  
 C1qr2s2  
•  Each   C1   molecule   must   bind   by   its   C1q   globular  
heads   to   at   least   two   Fc   sites   for   a   stable   C1-­‐
an8body  interac8on  to  occur  
•  When   pentameric   IgM   is   bound   to   an8gen   on   a  
target   surface   it   assumes   the   so-­‐called   “staple”  
configura8on  
Ø at  least  three  binding  sites  for  C1q  are  exposed  
•  Circula8ng  IgM  exists  as  a  planar  configura8on    
Ø the  C1q-­‐binding  sites  are  not  exposed  
Ø cannot  ac8vate  the  complement  cascade  

Santosh  Khanal  
Pentameric  IgM  in  planar  form  

Santosh  Khanal  
Staple  form  of  IgM  

Santosh  Khanal  
 C1qr2s2    
•  An  IgG  molecule,  on  the  other  hand,  contains  
only   a   single   C1q-­‐binding   site   in   the   CH2  
domain  of  the  Fc  
•  Firm   C1q   binding   is   achieved   only   when   two  
IgG   molecules   are   within   30–40   nm   of   each  
other   on   a   target   surface   or   in   a   complex,  
providing  two  a^achment  sites  for  C1q  

Santosh  Khanal  
 Forma8on  of  C3  convertase  
•  Binding   of   C1q   to   Fc   binding   sites   induces   a  
conforma8onal   change   in   C1r   that   converts  
C1r  to  an  ac8ve  serine  protease  enzyme,  C1r  
•  C1r  cleaves  C1s  to  a  similar  ac8ve  enzyme,  C1s  
•  C1s  has  two  substrates,  C4  and  C2  
•  The   C4   component   is   a   glycoprotein  
containing  three  polypep8de  chains  α,  β,  and  
γ  

Santosh  Khanal  
 Forma8on  of  C3  convertase  
•  C4   is   ac8vated   when   C1s   hydrolyzes   a   small  
fragment   (C4a)   from   the   amino   terminus   of  
the  α-­‐chain  
Ø expose  a  binding  site  on  the  larger  fragment  (C4b)  
•  The   C4b   fragment   a^aches   to   the   target  
surface  in  the  vicinity  of  C1  

Santosh  Khanal  
 Forma8on  of  C3  convertase  
•  The   C2   proenzyme   then   a^aches   to   the  
exposed  binding  site  on  C4b  
Ø the  C2  is  then  cleaved  by  the  neighboring  C1s  
•  The  smaller  fragment  (C2b)  diffuses  away  
•  The   resul8ng   C4b2a   complex   is   called   C3  
convertase  (converts  C3  into  an  ac8ve  form)  

Santosh  Khanal  
 Hydrolysis  of  C3  by  C3  convertase  
•  The   na8ve   C3   component   consists   of   two  
polypep8de  chains,    α  and  β  
•  Hydrolysis  of  a  short  fragment  (C3a)  from  the  
amino   terminus   of   the   α-­‐chain   by   the   C3  
convertase  generates  C3b    
•  A  single  C3  convertase  molecule  can  generate  
over  200  molecules  of  C3b  

Santosh  Khanal  
Hydrolysis  of  C3  by  C3  convertase  

Santosh  Khanal  
 Forma8on  of  C5  convertase  and  
Hydrolysis  of  C5  
•  The  C3b  binds  to  C4b2a  to  form  a  trimolecular  
complex  C4b2a3b,  called  C5  convertase  
•  The  C3b  component  of  this  complex  binds  C5  
and  alters  its  conforma8on,  so  that  the  C4b2a  
component  can  cleave  C5  into  C5a  
•  C5a  diffuses  away  
•  C5b   a^aches   to   C6   and   ini8ates   forma8on   of  
the  membrane-­‐a^ack  complex  

Santosh  Khanal  
Classical  pathway  of  complement  
ac8va8on  

Santosh  Khanal  
 Alterna8ve  pathway    
•  The   alterna8ve   pathway   generates   bound  
C5b,   the   same   product   that   the   classical  
pathway  generates  
•  But   it   does   so   without   the   need   for   an8gen-­‐
an8body  complexes  for  ini8a8on  
•  This  major  pathway  of  complement  ac8va8on  
involves   four   serum   proteins:   C3,   factor   B,  
factor  D,  and  properdin  

Santosh  Khanal  
Alterna8ve  pathway  contd.  

•  The   alterna8ve   pathway   is   ini8ated   in   most  

cases   by   cell-­‐surface   cons8tuents   that   are  
foreign  to  the  host  
•  Both   Gram-­‐nega8ve   and   Gram-­‐posi8ve  
bacteria   have   cell-­‐wall   cons8tuents   that   can  
ac8vate  the  alterna8ve  pathway  

Santosh  Khanal  
 Hydrolysis  of  C3  
•  Serum   C3   contains   an   unstable   thioester  
bond,   is   subject   to   slow   spontaneous  
hydrolysis  to  yield  C3a  and  C3b  
•  The   C3b   component   can   bind   to   foreign  
surface   an8gens   (such   as   those   on   bacterial  
cells  or  viral  par8cles)  

Santosh  Khanal  
 Forma8on  of  C3  convertase  
•  The  C3b  present  on  the  surface  of  the  foreign  
cells   can   bind   another   serum   protein   called  
factor  B  to  form  a  complex  stabilized  by  Mg2+  
•  Binding  to  C3b  exposes  a  site  on  factor  B  that  
serves   as   the   substrate   for   an   enzyma8cally  
ac8ve  serum  protein  called  factor  D  
•  Factor   D   cleaves   the   C3b-­‐bound   factor   B,  
releasing   a   small   fragment   (Ba)   that   diffuses  
away  and  generates  C3bBb  

Santosh  Khanal  
Forma8on  of  C3  convertase  contd.  

•  The   C3bBb   complex   has   C3   convertase   ac8vity  

and  thus  is  analogous  to  the  C4b2a  complex  in  
the  classical  pathway  
•  The  C3  convertase  ac8vity  of  C3bBb  has  a  half-­‐
life  of  only  5  minutes  unless  the  serum  protein  
properdin  binds  to  it  
•  Properdin  stabilizes  it  and  extends  the  half-­‐life  
of  the  convertase  ac8vity  to  30  minutes  

Santosh  Khanal  
 Forma8on  of  C5  convertase  
and  Hydrolysis  of  C5  
•  The  C3bBb  generated  in  the  alterna8ve  pathway  
can   ac8vate   unhydrolyzed   C3   to   generate   more  
C3b  
•  The   C3   convertase   ac8vity   of   C3bBb   generates  
the   C3bBb3b   complex,   which   exhibits   C5  
convertase   ac8vity,   analogous   to   the   C4b2a3b  
complex  in  the  classical  pathway  
•  The  nonenzyma8c  C3b  component  binds  C5,  and  
the   Bb   component   subsequently   hydrolyzes   the  
bound  C5  to  generate  C5a  and  C5b  
•  C5b  binds  to  the  an8genic  surface  
Santosh  Khanal  
Alterna8ve  pathway  of  complement  
ac8va8on  

Santosh  Khanal  
 Lec8n  pathway  
•  Lec8ns   are   proteins   that   recognize   and   bind   to  
specific  carbohydrate  targets  
•  Also   known   as   MBL   pathway   or   mannose-­‐binding  
lec8n   pathway   because   the   lec8n   that   ac8vates  
complement  binds  to  mannose  residues  
•  The  lec8n  pathway,  like  the  alterna8ve  pathway,  
does  not  depend  on  an8body  for  its  ac8va8on  
•  However,  the  mechanism  is  more  like  that  of  the  
classical  pathway  
•  Aler   ini8a8on,   it   proceeds,   through   the   ac8on   of  
C4  and  C2,  to  produce  a  C5  convertase  
Santosh  Khanal  
Lec8n  pathway  contd.  

•  The  lec8n  pathway  is  ac8vated  by  the  binding  of  

MBL   to   mannose   residues   on   glycoproteins   or  
carbohydrates   on   the   surface   of   microorganisms  
including:  
Ø Salmonella  
Ø Listeria  
Ø Neisseria  
Ø Cryptococcus  neoformans  
Ø Candida  albicans  
•  Its  func8on  in  the  complement  pathway  is  similar  
to  that  of  C1q,  which  it  resembles  in  structure  

Santosh  Khanal  
Lec8n  pathway  contd.  

•  Aler   MBL   binds   to   the   surface   of   a   cell   or  

pathogen,   MBL-­‐associated   serine   proteases,  
MASP-­‐1  and  MASP-­‐2,  bind  to  MBL  
•  The  ac8ve  complex  formed  by  this  associa8on  
causes  cleavage  and  ac8va8on  of  C4  and  C2  
•  The   MASP-­‐1   and   MASP-­‐2   proteins   have  
structural  similarity  to  C1r  and  C1s  and  mimic  
their  ac8vi8es  

Santosh  Khanal  
 Membrane-­‐A^ack  Complex  
•  The   terminal   sequence   of   complement  
ac8va8on  involves  C5b,  C6,  C7,  C8,  and  C9    
•  I n t e r a c t   s e q u e n 8 a l l y   t o   f o r m   a  
macromolecular   structure   called   the  
membrane-­‐a^ack  complex  (MAC)  
•  MAC   forms   a   large   channel   through   the  
membrane  of  the  target  cell  
•  Enable   ions   and   small   molecules   to   diffuse  
freely  across  the  membrane  

Santosh  Khanal  
Membrane-­‐A^ack  Complex  contd.  

•  The   end   result   of   ac8va8ng   the   classical,  

alterna8ve,  or  lec8n  pathways  is  produc8on  of  an  
ac8ve  C5  convertase  
•  C5   convertase   cleaves   C5,   which   contains   two  
protein  chains,  α  and  β  
•  Aler   binding   of   C5   to   the   non-­‐enzyma8c   C3b  
component   of   the   convertase,   the   amino  
terminus  of  the  α  chain  is  cleaved  
•  The   cleavage   generates   the   small   C5a   fragment,  
which  diffuses  away  
Santosh  Khanal  
Membrane-­‐A^ack  Complex  contd.  

•  The  large  C5b  fragment  binds  to  the  surface  of  

the  target  cell  
•  Provides   a   binding   site   for   the   subsequent  
components  of  the  membrane-­‐a^ack  complex  
•  The   C5b   component   is   extremely   labile   and  
becomes   inac8ve   within   2   minutes   unless   C6  
binds  to  it  and  stabilizes  its  ac8vity  

Santosh  Khanal  
Membrane-­‐A^ack  Complex  contd.  

•  As   C5b6   binds   to   C7,   the   resul8ng   complex  

undergoes   a   hydrophilic-­‐amphiphilic  
structural  transi8on  
•  Exposes   hydrophobic   regions,   which   serve   as  
binding  sites  for  membrane  phospholipids  
•  On   a   target-­‐cell   membrane,   the   hydrophobic  
binding   sites   enable   the   C5b67   complex   to  
insert  into  the  phospholipid  bilayer  

Santosh  Khanal  
Membrane-­‐A^ack  Complex  contd.  

•  Binding   of   C8   to   membrane-­‐bound   C5b67  

induces  a  conforma8onal  change  in  C8  
•  Undergoes   a   hydrophilic-­‐amphiphilic  
structural   transi8on,   exposing   a   hydrophobic  
region,   which   interacts   with   the   plasma  
membrane  
•  The  C5b678  complex  creates  a  small  pore,  10  
Å  in  diameter  

Santosh  Khanal  
Membrane-­‐A^ack  Complex  contd.  

•  The   binding   and   polymeriza8on   of   C9,   a  

perforin-­‐like  molecule,  to  the  C5b678  complex  
results  in  the  forma8on  of  MAC  
•  As   many   as   10-­‐17   molecules   of   C9   can   be  
bound   and   polymerized   by   a   single   C5b678  
complex  
•  During   polymeriza8on,   the   C9   molecules  
undergo   a   hydrophilic-­‐amphiphilic   transi8on,  
so  that  they  too  can  insert  into  the  membrane  

Santosh  Khanal  
Membrane-­‐A^ack  Complex  contd.  

•  The   completed   MAC   has   a   tubular   form   and  

func8onal  pore  size  of  70–100  Å  
•  Consists  of  a  C5b678  complex  surrounded  by  a  
poly-­‐C9  complex  
•  The   ions   and   small   molecules   can   diffuse  
freely  through  the  central  channel  of  the  MAC  
•  The   cell   cannot   maintain   its   osmo8c   stability  
and   is   killed   by   an   influx   of   water   and   loss   of  
electrolytes  

Santosh  Khanal  
Membrane  a^ack  complex  

Santosh  Khanal