Download Low resolution scan

Burr
BIOL 4345/6351
Immunobiology
(Former Chapter 2, 1st & 2nd Editions)
Chapter 4: Antibody Structure and the Generation of B-cell
Diversity
(Notes for the lectures covering Chapters 1 & 3 were passed out in the first class) (Chapter 2
will be covered in subsequent lectures.)
Plasma cells secrete antibodies of the same antigen specificity as the
membrane-bound immunoglobulin expressed by their B-cell precursor.
• “Antibodies are variable antigen-specific proteins produced by B lymphocytes
of the immune system in response to infection.”
• “Antibodies are the secreted form of proteins known more generally as
immunoglobulins (Ig).” Each of the trillions of B-cells present in the body at
any given time can make only one particular kind of immunoglobulin, and
before it has encountered antigen, a mature B-cell expresses that one particular
immunoglobulin on its cell surface. If that B-cell happens to encounter its
cognate antigen during its lifetime, binding of the antigen to the surface
immunoglobulin stimulates the B-cell to proliferate and differentiate into a
plasma cell. The plasma cell then secretes antibodies of the same specificity as
that of the membrane-bound immunoglobulin (Fig 4.1). Antibody production is
the single effector function of B-cells.
IgM
Interleukins
TH2
cell
33(b)
Secreted
IgM
(or IgG,
IgA, IgE,
etc.)
The Antibody Molecule
(Immunoglobulin G in this example)
• Secreted immunoglobulins (“antibodies”) are glycoproteins that are built from a
basic unit of 4 polypeptide chains: 2 identical heavy (H) chains and 2 identical
light (L) chains, assembled into a structure resembling the letter Y (Fig 2.2, left
panel). The heavy and light chains are held together by disulfide bonds.
Carbohydrate is attached to the heavy chains. The amino termini of both the
heavy and the light chains are located at the top of the Y structure.
• The polypeptide chains of different antibodies vary greatly in amino-acid
sequence, but all the sequence differences are concentrated in the amino terminal
region of both the heavy and light chain; this region is known as the variable
region or V region. This variability is the basis for the great diversity of
antigen-binding specificities among antibodies, since the paired V regions of a
heavy and a light chain form the antigen-binding site (Fig 2.2, right panel).
• The remaining parts of the light chain and the heavy chain have a much more
limited variation in amino acid sequence, and are known as the constant regions
or C regions.
Two identical antigen-binding sites
33c
The Immunoglobulin molecule can be dissected by proteases
In IgG, a relatively unstructured region in the middle of the H chain forms a flexible
hinge region, that can be cleaved by the protease papain to produce 3 fragments: 2
Fab fragments (Fragment antigen binding) and one Fc fragment (Fragment
crystallizable) (Fig 2.3).
(Janeway, 6th Ed, fig 3.3; equivalent to Parham fig 2.3)
Papain:
disulfide
bonds
Pepsin:
(In your Parham text [fig 2.3], the illustration incorrectly indicates that papain cuts C-terminal to
the pair of disulfide bonds in the “neck” of the immunoglobulin molecule; in fact, it cuts Nterminal to the disulfide bonds as shown in the figure above, taken from the Janeway text.)
33 (d)
Differences in the heavy-chain constant regions define the 5 main
isotypes or classes of immunoglobulin: IgG, IgM, IgD, IgA & IgE
• The heavy chain of each of these isotypes is denoted by the corresponding lower
case Greek letter (α, δ, ε, γ, µ).
(Pentamer)
(Secreted
as dimer)
(Expressed by mature, naïve B cells)
(There are only two types of light chains: κ andλ; the choice is random and there are no functional
differences in the immunoglobulins containing either a κ or a λ light chain.)
• IgG: most abundant antibody in blood; functions best in opsonization. (“Opsonization” =
coating of pathogen by antibody molecules, which makes it easier for macrophages to
ingest pathogen.)
• IgM: found on surface membrane of mature, naive B-cells; released in soluble
(pentameric) form when B-cells mature into “plasma cells”; first isotype made in
response to pathogen; best at activating complement.
• IgD: Minor immunoglobulin; no special function known; expressed as surface
immunoglobulin by mature, naive B-cells; expression ceases after antigenic stimulation.
• IgA: Secreted onto internal mucosal surfaces, and into mother’s milk; more IgA is
synthesized by the body than any other isotype; IgA is secreted as a dimeric molecule.
• IgE: fights off parasites (worms, helminthes, etc); is bound to the surface of mast cells,
which underlie both skin and internal epithelial surfaces.
B-cells start off making surface IgM (and IgD); in response to binding of their
cognate antigen, they differentiate into plasma cells, secreting pentameric IgM.
Later, they usually switch (“isotype switching”) to making (and secreting) either
IgG, IgA or IgE; IgG-secreting plasma cells can later switch to making IgA or IgE.
33(e)
The heavy and light chains of an immunoglobulin molecule are made
from a series of similar protein domains, called the
“immunoglobulin domain”
• Heavy and light chains both consist of a series of similar sequence motifs; each such
motif consists of about 100-110 amino acids, and is called an “immunoglobulin
domain.” There are 4 such domains in the IgG H chain: 3 constant (CH1, CH2, &
CH3) and 1 variable (VH). The light chain has 2 such “immunoglobulin domains”:
CL and VL
Immunoglobulin domains
33 f
The three-dimensional structure of immunoglobulin C and V domains:
• The structure of the immunoglobulin domain resembles a bulging sandwich, in
which the 2 slices of “bread” consists of a set of β sheet strands interacting side by
side (as all β sheets by definition do) by peptide backbone hydrogen bonds. The
“filling” of the sandwich is the hydrophobic side chains of the β sheet amino acids.
The structure is further stabilized by a disulfide bond between the two β sheets.
Many other proteins (particularly cell-surface and secreted proteins) have
subsequently been found to contain regions of repeating immunoglobulin-like
domains; such proteins collectively form the immunoglobulin superfamily.
Polypeptide chain
Anti-parallel Beta
sheet structure
side chains
Anti-parallel Beta
sheet structure, viewed
from the side.
Anti-parallel Beta
sheet structure,
viewed from above
Hydrogen bond between
carbonyl oxygen : amide NH
33g
The antigen-binding site of an antibody molecule is located in what
are termed “hypervariable” regions of the heavy-chain and lightchain V domains:
• A comparison of the V domains from many different antibody molecules reveals
that the almost all the amino acid differences are concentrated in 3 regions on
both the heavy and light chains. These particularly variable regions are called
the hypervariable regions. On the heavy chain they are referred to as HV1,
HV2, & H21; on the light chain, LV1, LV2, LV3.
• Another name for these hypervariable regions is complementarity-determining
regions (CDRs).
• These 6 hypervariable regions (3 on the heavy chain, 3 on the light chain) form
the antigen- binding site on the antibody molecule
“FR” (FR1, FR2, etc) = Framework Regions
33 h
The part of an antigen to which an antibody binds is called an
“antigenic determinant” or an “epitope”
• Antigenic proteins typically contain several different epitopes (antigenic
determinants); epitopes for proteins typically consist of several amino acids
located on the surface of the protein.
Fig 2.8 below shows the coat of poliovirus, which consists of three different proteins
(shown in yellow, blue and pink). Known epitopes of the virus are shown as white
patches. One of the viral proteins (VP1) (blue) is shown separately, but folded as it
is in the virus particle. This protein contains several different epitopes (white),
located on the surface of the protein, and exposed on the surface of the virus
particle.
Whole polio
virus
White = epitopes on the
blue viral capsid VP1
protein
Polio virus
capsid protein
33 i
Any antigen that contains more than one epitope (Fig 2.9, left panel),
or more than one copy of the same epitope (right panel) is known as a
multivalent antigen.
33 j
Epitopes
The part of an antigento which an antibodybindsis calledan antigenic
determinant oI an epitope. In naturethesestnrcturesareusuallyeither
carbohydrate
b?proteinor both.
" Someepitopesarelinear(panela), othersarediscontinuousor "conformational"(panelb).
Hs'e'rt
?
Crrfrn
I t
rt.*
I
. L
t.tl t.y.
3ffi-ffiArtqmiiflrr
- ,.t'**' 't '\'*o\*rta 'I o.\**-\ "1'\'yo'
N rrt p-.4^'
f
t o r * X L o u , * tU Y * * \ " V o l i o ' '
3rf (b)
eo^^b,;J
E fi h f " '
,.,.tonJJ
g,\'
,ua $^".,
r
r
t,^J,;; , 't"' " F o^'ftL" JtLs:
'1,^ff*
dkis
q
S$uw*.$S
t
ffq-4$0".
{$' lgr ${'
$*)*''''T
@-.1
t-t4^{
*b
Wr
..
f,{'
A"*u
1.
,1
{
*
1
r,[r-rf "n!;hwmsrrhlffi**igntlryffi**sffiffi
. ;*
&
i,$ rs\
$ t
, ,qs,tl' *t
{\*'t'r*"*$ lu r* , \# d'! *air1'nr
Y
*Ls\seRg
ww*
'
he*4^\1 *-T':$qii
' ' n ry*d, :
',,
"'i"':'
i'1i'.
s
rlln ?,.iu*al;l
*$l'' ,il_
|
' 4 " , * l \ 1 *l ;
*W
---
. u**fu B**$\ yb-\
ffiffit-H-{d$**
i*:s
i*strr},*st{ihq.:*e;$.
\,,{{-}t"lt-}*
db.4f
&ry
$n ti:*: i.:ilst-ti:* olni--'lt'illi'*I'rt:*$til:E
*l:trh*.r{ii*Sir.ri}tri\rL$rrlr,rtiti i*:g, l't.:*^
l \\ ils t{.:
i,ti:ti-s::;lk*t'u:t':cllll
;t iitr$i*: ill'li rti* i t $r.:*l"
sn*s:i;,t"*,
i:"sr
F
;-&.\
ft-T-fw*e*'*t{*
&{s * *stt }{*o
Wffi
ffi ffiw
h*r;.i* ]. nh rn hi**.eJlt *l t"*-Ur;i;+r
tlr.:tlhi*i* *ntis rlir}l {r+iti*:it
ilit*r"un'{li:;
ltr:di*ls3.
is ll iy'*ixtt.il.* r.rl' ltsi-{i
*{ .ffees*ss'**
S*otts
*
f*&*b h* w* !*nF
fL* nblt;$s&*
w**k* ***itu**g3
s$
....i
"."-'...,,.".........
h'$r.r* *l* I*:ii,*I ;tyl*i hr-lrl;l'[ * * hnr.:itr$'\'
*:*,*:iit rt,utk,*i*: lirltri*ri{'*:*X
p:r*is",r:tits
* *t,tt* * i tr.*ti h*::eii * s it I'
s*g.:3:
I-v,r.ri- i 13
i fi*:i,tvk *r*lr+'t:s,1:*,*
t_"_,*
{*"t
* ksk*i $ y&k
.l
*mw**try &* &w-6
*#w s*st.&
sf*J*u,w*
,r+"Ai^*'
Cl-".*,!6*
? tl*{..rtJ
t
$ee
\\
r'
/vft!'
B c"\\'
ef € lw**e*w\
**[*
ft(,
\fr
f.: \
[-kqeRrq
*----J
w. 'Swwffie
stu4ffitu
"klor f
'l."
5"1
hr\bortf .. 'I
i\ii
el"\\,
h*gLr,-J
i
*r*fit*s,$eiit l*ttd* *usgr*il.$'um*,**
**t*$s*,*d xn{*"l***,i+''ffifffiT*
m*,sWW$s,ffi,ft
'
m:-1u,*t**rm*',*:l**';*i'*
''*
i;' G": *'L't'
\"f.'
i b*t
gftfn^-J;,J'
q"etBru 4
*
lr"\
l;:fit'!'**')
-:ill
\waa.1*"'[::{5':*
g*ef
$
"
tw
*,**
tr
m**ry
i l S & w S W . & * * *"b. 3
F t
€
_*****#
r
*q *sk** %;Sd . t
-k
#.*$4
g \
cffi
ffiffiffiffiffiffi
& & & & # % &
$xr*
llii *.hvn'6
ifi:.lSS.$il $xw'a'
i*r #*el*****r* mr$mrr*f
ffifu-qe?
qfcd n* *,
t4 *f
J i., lecdn*o,s('tr"r
f e&oP"I
a * . <* G
&c&
Hef s.tr
g,p*eww
W,*s$.. g
- tl* [,^r"J l\ 1".\o'*o
C.etLlto * \kftf
r''i
e
**o
**r* 6tq***
One contribution to the diversity of antibodies comes from the random joining of V, D, and J
segments to create genes for immunoglobin heavy chains (H), and light chains ( and ).
(35)
Fig 4.18 (3rd Ed)
(Replaces fig 2.16
from 2nd Ed.)
(30)
(40)
(4)
.
35 x 5
= 175
30 x 4
= 120
175 + 120
= 295
40 x 23 x 6
= 5,520
295 x 5,520
6
= 1.6 x 10
For the human Kappa light chain, approximately 35 VK gene segments and 5 JK
segments can be recombined in (approximately) 175 (35 x 5) different ways.
For the human Lambda light chain, approximately 30 V gene segments and 4 J
segments can be recombined in (approximately) 120 (30 x 4) different ways.
So a total of approximately 295 (175 + 120) possible light chain genes ( plus )
could be created by recombining V and J segments
For the human Heavy chain, approximately 40 VH gene segments, 23 D segments, and 6
JH segments can be recombined to make approx 5,520 (40 x 23 x 6) heavy chain genes.
So if recombination between different gene segments were the only mechanism of
producing the diversity of antibody genes, then approximately 1.6 x 106 different kinds
of antibody genes could be created by this mechanism alone (295 Light chain genes x
5,520 Heavy chain genes).
39
An additional way of generating diversity: The recombination process that
joins V-J and V-D-J segments is “error prone”:
Each V, D, or J gene segment is flanked by a repeat sequence called a “recombination
signal sequence” (“RSS”). There are two types of RSS: RSS1 (7-23-9) & RSS2 (9-12-7).
RSS1 and RSS2 are inverted with respect to each other.
(Spacers vary in sequence, but have fixed length)
23 nucleotide “Spacer”
12 nucleotide “Spacer”
7 = “heptamer”
9 = “nonamer”
RSS1
RSS1
RSS2
RSS2
RSS2
RSS1
X
(No V-J joint for H chain [always V-D-J] because
can’t pair up RSS1 with another RSS1
In the recombinational cut and join event that joins a V segment with a D or J segment,
an RSS1 sequence pairs with an RSS2 segment. This pairing, and the subsequent cutting
and joining, is mediated by enzymes called “RAG-1” and “RAG-2”, acting together with
some other general purpose DNA modifying enzymes.
(“RAG” = Recombination
Activating Genes)
1
1
2
2
1
2
1
2
Other cellular general purpose
recombination enzymes
40
(".&*"p'..J'lrl."l..[lls
Sllo? t.
FUEI|#
D .Jl i (..1.. 4
orfGt.ll I ir tr. i l1
(,notl* l*{ El:t,e*)
::,Hilfikr il
::,,-.',111.i,:.15
{r
offiGdrldPr,nQn"ffiiffseu
.[Jf '.
ti'J:
\
ilfn.nt,.tryf
.t-")d r\|.l,y
;::":'i;ilt.t ) 4rrr):
::::.::Fiii$*;:
lr:')
FWtt€{i
$.* -.)."
o*tti
l't
Fheplaml --l
\\o
t$.';.
Y*rl'rrr*'
n*.il'J
c " .c c \ c
\ott'
(9"\,iJ r"^i")
4f'
( N"*- Xer,,^\i'nc)
\ !"o*'r'u{'
,-<..i.":)
L
n<*t
(81999 ElsBViBrScience/GarlandPublishing
.? t: t.ol]j."J .
bc irr<--..i..r.
.i-
ht(ut err.*Iltl.t
i'
II92 I CHAPTER THIRTY-SEVEN
(rr. GcGro..Xr.^..{ Lt,l1. ^L....
tecr gror.lcf
I
Several proteins that may be associated
with V(D)J joining have been characterized,
including heptamer binding, nonamer binding,
and endonuclease activities. The onlv case in
which there is direct evidence for involvement,
however, is that of the &{G genes. RAG( and
MG2 are two genes, separated by <10 kb on the
chromosome, whose transfection into fibroblasts
causes a suitable substrate DNA to undergo the
V(D)J joining reaction. This suggests that their
products either directly form a recombinase or else
are able to activqte an enzyme that is ubiquitous
(but is inactive in their absence). Mice that lack
elther MG I or RAG2 are unable to recombine their
immunoglobulins or T-cell receptors, and as a
result have immature B and T lymphocytes.
What is the connection between joining of V and
genes
C
and their activation?Unrearranged V genes
are not actively represented in RNA. But when
a'V gene is joined productively to a C* gene, the
resulting unit is transcribed. However, since the
sequenceupstream of a V gene is not altered by the
joining reaction, the protnoter must be the same in
unrearr&nged, nonproductiaely rearcanged, and
p r oductiu ely rearranged genes.
A promoter lies upstream of every V gene, but is
inactive. It is activated by its relocation to the C
region. The effect must depend on sequences
downstream. What role might they play? An
enhancer located within or downstream of the C
gene activates the promoter at the V gene. The
enhancer is tissue-specific; it is active only in B
cells. Its existence suggeststhe model illustrated in
Figure 57.15, in which the V gene promoter is
activated as soon as it is brought into reach of
the enhancer.
Each B cell expresses a single type of light chain
and a single tlpe of heavy chain, because only a
single productive rearrangement of each type
occurs in a given lymphocyte, to produce one light
and one heavy chain gene. Because each event
involves the genes of only one of the homologous
chromosomes, the alleles on the other chromosorrre
are not erpressedinthesamecell This phenomenon
is called allelic exclusion.
The occurrence of allelic exclusion complicates
the analysis of somatic recombination. A probe
reacting with a region that has rearranged on
one homologue will also detect the allelic
sequences on the other homologue. We may
brlrEsft lnb thegoximttyof anqrlrqfrcsrIntheC
god€.Th€enhancsrls acth€onlyIn B tymphorytes.
Enhoncer
rt.
r . t . : 1' ,1, : u i l ! i {
.'
,,filli,
'rlllll i'
ililf
,Jlll[,,
't
: t , i : i : l r : i t ,. i"
I
l:G.:f.f. |.[ q!ts
,'
:,i,
] , : r, r r , r r ir'ri
\nilt
v
a
} Y
t
'Pronioter
l
t-
r I
b
E
Enhoncer
l
l
l1
9, ryr{erit lh ertt-tl.r $e.r qn
( S\hrbrrJ )
9'\l
ruftffi
$ffiic,,
rucilnbigtkn
RlmryRin
J .f,^* lt
- c o d1 r $, 1
t"
(tu
su"tct'
",1
i
",
"
i s ho s g\
'
t*o^ C \o tro^ ot"' it
A\
Ozm0GtrfdFllC*rylEmlr$rm
5eyv,u*-
{
The B-cell receptor:
• B cells express membrane-bound immunoglobulin M in association with two other
proteins: Igα and Igβ
• Igα and Igβ have cytoplasmic tails that associate with other proteins, to mediate the
signal-transduction events leading to endocytosis, growth, and differentiation.
Ag
Ag
Transmembrane IgM
Signal transduction proteins
associate with the cytosolic tails
of the Igα and Igβ proteins.
Signal transduction events, leading to
proliferation and differentiation of Bcells into plasma cells.
Igα and Igβ join the immunoglobulin molecule in the lumen of the ER. This association is
required for export of the complex from the ER to the cell surface.
47
Rearranged V-region sequences are further diversified
by somatic hypermutation:
• Once a B cell has been activated by antigen, it begins to proliferate. During this
proliferation, there is a high rate of error when DNA polymerase replicates the Ig Vregions, leading on average to about one mutation per V-region sequence per cell division
(million fold greater rate than normal). Process is called somatic hypermutation.
• Somatic hypermutation gives rise to B-cells expressing mutant Ig molecules on their
surface; some of these mutant Ig’s will have a higher affinity for the antigen, and these B
cells will preferentially proliferate and mature into plasma cells. This phenomenon is
called affinity maturation.
“Antibodies raised by immunization with the same antigen were collected one and two
weeks after immunization, and their AA sequences were determined. Each line represents
one sequenced antibody, and the red bars represent AA positions that differ from the
prototypic sequence.”
48