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UNIVERSITY Of JORDAN

Faculty OF Medicine

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LECTURE NO:

,\----1 DATE: 22,/9 /zoo

1+---1 DONE BY: Ha. 'j a.. ,


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Lecture #:4

Immunoglobulins (antibodies):
We discussed previously the molecular basis of antibody specificity; how
the specificity of the antibody is determioed ? what part of the molecule
determines the specificity and how the antibodies diversity is produced??

Diversity: is the preseoce oflarge oumber (millioos) of differeot types of


antibodies.

Antibodies are formed of two light chaios and two heavy chains.
Example:
IgG
The oumber of amino acids in the light chain is about 214 amino acids
The oumber of amino acids in the heavy chaio is about 446 amioo acid
( which is twice as much as the light chain)

5-5- - $ _5

As the figure above shows :


Heavy chaios are coooected together by disulfide boods • and each light
chain is booded to the heavy chain by disulfide bood.
The cleavage with papain (that produce partial digestion) produce half of the
two heavy chains as Fc fragment and two Fab fragments.

The amino acid sequence of the light chain like any amino acid sequence
requires purified protein we cant study the amino acid sequence on a
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mixture of antibodies, we need one type of antibody (multiple myloma).

Q One type of antibody can be obtained from a patient sufffering from


multiple myloma ; malignant growth of one single plasma cell. Thus all the
antibodies produced in these cancer cells will have the same type and
therefore the same amino acid sequence!

Amino acid sequence of the light chains of a multiple myloma patient show
that the amino acids from 1-108 vary (differ) from one patient to the other.
i.e: for each patient there is a unique sequence of the amino acids from the N
terminal to almost half of the light chain.

N~"======--~- C
'0'
whereas the amino acid sequence from 109- 214 is the same in many patient
( not necessary exactly the same in all patient. we expect a norrnallittle
variation in amino acid sequence from one patient to another)

we conclude that there are two regions:


I-variable region (because the amino acid sequence vary from one type of
antibody to another)
2-constant region
( half the molecule is variable and the other halfis constant)

Within the variable region ( 1-108) , it was found that there are streches of
amino acids that shows prominent variability ( the sequence vary greatly
from one patient to anotheF::> hypervariability ) , where there are some
regions that show less variahlility.

The heavy chains which are formed of 446 amino acids, also have one
variable region oflbe same length (108 a.a) that have a unique sequence
which varies from one patient to the other. The rest from 109-446 are
similar in many patients .

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So both light and heavy chains have variable and constant regions!!

Note that:
the variable region of the light chain (V L) equals in length to the variable
region of the heavy chain (Vh)·
V L= V h

The constant region of the heavy chain is almost three times the constant
region of the light chain.
Ch = 3 • CL
So the variability is more in three segments of the variable region: these are
known as hypervariable segments.

The function of these hypervariability segments is probably these are the


segments or a.a sequence where antigen binds.
It means they form antigen binding sites and this was proved by a technique
called Xray crystallography.

What is Xray crystallography???


It is a technique that
1- shows the three dimensions of the protein molecules.
( in it we convert protein to protein crystals)
2- determine the location of every a.a in the three dimension.

It was found in Xray crystallography that the hypervariability regions are the
binding site for the antigen. Therefore they were named Complementarity
Determining Regions ( CDR).

Complementarity Determining Regions ( CDR):


These are the regions which are complementary in shape to the antigen.
Binding an antigen to the antibody is like the binding of a substarte to an
enzyme. They fit together like key and lock ( they complement each other).

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In the figure above note that in the light chain the length of the variable
region is almost the same as the length of the constant region.
Where the constant region of the heavy chain is found in three domains and
it is three times in length as the constant region of the light chain.

The three dimensional structure was studied for the variable and constant
regIOns:

Two b J Sh.oeb
iL>f a,..,hF- II .. 1 ~
Sl-,..",... J.~

This shows us the shape of the constant domain and variable domain in the
light chain.
This represents Bsheets as if you have two layers of Bsheets together and
there are loops that connect the streches of the Bsheets.
The Complementarity Determining Region is the region that connects one B
strand with the other.
The variable region contain loops where the antibody binds the antigen.

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[fthe number of the anribodies is huge (millions) :
How can the nucleotide sequence in the gene produces this large number of
peptides (antibodies).
Are there million of genes that produce these million of antibodies??
Actually NO, because the antibody specificity is dtermined by the light
chain sequence and the heavy chain sequence.

Example:
If we have 100 kinds of light chains and 100 kinds of heavy chain, when
they combine in a random way knowing that we can combine every heavy
chain with every light chain, we will have
100 • 100 = 10000 molecule
This is one way of how we can get a large number of antibodies; every cell
will produce one type of light chain and one type of heavy chain and they
combine in different cells to produce different antibodies!

This is not the only way:


The production of proteins from a gene occurs through this flow chart of
infonnation:

1
1
o_
J
o
---7"--- -. RillA
i
fro. .... .,I.+~_ ..... •• ..... RNA
P.I,:3,. ... f/""',..... s-::3~""''''S''3

1- Double helix of DNA( this is a gene of a protem)


2- Synthesis of mRNA by transcription
mRNA contains the sequence of nucleotides that produce the polypeptide
chain but in many mRNA types, its sequence may contain regions that are
not translated.

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3-How can we get rid of it??? By splicing.
i.e: The part that is not expressed will be removed by this process (splicing)
and the other expressed regions will be joined again.
4- muture mRNA that will be used for translation.
(Here we are talking about one gene producing one peptide)

In the case of antibodies and during cell differentiation, there will be


rearrangement of the gene.

[fthere is different sequence from 1-[08 and the sequence is the same from
[08-214 (regardless of specificity), by logic there is the same gene in every
antibody otherwise we expect the constant region to vary from one kind of
antibody to the other. So this part of the polypeptide is determined by just
one segment of the gene.

There are multiple V genes and only one C gene (one gene for constant
region).

During development when the e,mbryonic cells convert to differentiated


plasma cells, one of these V genes will join with C region gene, to give us
one whole gene that is variable with constant.
(Y'H.A.. I~;f'k V =S"""''''=' one. C .,_..,e.
v ... V .. I
_____ .\1....

cl: rrc.rt.'"""" -.k J. vc


1
c~ll

During differentiation of the cell only one segment of the V region will be
chosen and this will be joined with the C region.

They found that the genes of the variable regionare numerous.


The V genes in the embryonic cells encode 95 a.a ( the V genes do not
encode 108 a.a, 95 or 97 only!)
The 13 a.a that are left, from where they come??
They come from another segment called J genes ( J stands for joining,
joining V with C ).

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Gene is present in three segments VJC (comple gene) with different
combinations.

Example:
V, to V100
J,toJ,
C,

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~l" I[

V2J4 are joined ,the rest are removed not by splicing but it is on DNA level
(gene rearrangement)
~
Only one gene of V segment will join only one gene of J segment as we
have in the above example:
We have
40 V genes
5 J genes
so 40 • 5 ~ 200 possibilities of genes for the light chain.
Then transcription ofmRNA followed by splicing that will remove the
sequence between the VJ and the C --. light chain.

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,....

1- [=r"1111
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rJGUll n.14 1M • qtrt..cfWn Ious. 11111; pat of Iunin chon_
..., of 4O..-as!hlt.-dr n ..... M ~ (~Iftd
of til. lip dwin, .., ~. 5 ~ N ..-dr 1he ;o;n;.., (ll1ql
.-110). Mtd . . . . rqion!hlt encadl!$lhe CllfI5Wot {Q..pon.

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flCiURf »'16 Llpt'<hin up,"sIo",
Th. ~-<NIn prottin Is upmgd by
lI'anscrf~ cl the _,..td feI'W: to
proclK.e • ~NA ~ with h '"
llncl C rtJionl .. p.mted. RNA spIdt1(
~ dM _.-... MqUC"nceII to
prntto...... - "'ItNA ~ with the VI
.nd C ~Ifons lin",", nlf\Htion 01 the
mfl.NA Ind prouuiltJ of the Initial prottln
ptOdl.Kt proctucfl the IIfht <.hIm..

How the Ig class change from IgM to IgO , or from IgM to IgO???
All we need to change is the gene of the constant region in the heavy chain
.The gene tells wether the heavy chain is M,O, E or A type.

Changing the constant region of the heavy chain datermines what is the class
of the antibody IgM, IgO, ofIgA.
In this case only the class of the antibody is changed but the specificity is
not.

Note:
What the doctor explained about the light chain implies on the heavy chain
as well, adding that there is a D segment.

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--+ The body produces initially IgM type then change it to IgG without
changing specificity (variable region stays the same, we only change the
constant region.) we change the class from IgM to IgG.

Bioenergetics

Bioenergetics: is the study of energy In the living systems.

Life reqiures both energy and matter.


We eat or need nutrients In order to sustain life. Cellularly we
need energy in order to sustain life.

The energy Is very Important to the cell.


No energy, no life.

The cause of death Is almost loss of energy.


Example:
Hemorrhage:
Severe blood loss (bleedlng)that means no blood is reaching to
the brain so the brain cells cannot produce adequate energy.
Myocardial Infarction will occur, arteries will be blocked, no more
O2 will reach the cells.

Energy is one of the major purposes of nutrients (nutrients are for


energy).

Why energy Is needed??


1- mechanical work: includes
a) muscular contraction
b) beating of flagella and cilia
c) movement of chromosomes during cell division.
d) any motion requires energy.

2- active transport:
the cells cannot stay alive without active transport.
Nerve cells work by active transport I constantly there is
pumping of sodium outside the cell I and potassium to the Inside
of it.

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The function of the cell depends on this membrane potential that
Is achieved by active transport which requires energy. Also,
absorption of nutrients (absorption of glucose through intestinal
cells) requires energy.

3- biosynthesis:
synthesizing large molecules from small monomers requires
energy.
Ex:
Proteins are produced by joining amino acids.

4- heat (not the least important):


It Is produced as by-product in all of the above cases.
Active transport needs energy, energy conversion Is not 100% I
some of the energy will be liberated as heat .The liberation of
heat Is useful ,it keeps the body warm even In cold climates.
Sometimes, heat production in newborns is extremely important
to keep the baby warm.

Sun is the ultimate source of energy for all living oraganisms.


Even our body depends on the sun's energy.

How come???

Flow of energy (unidirection):


:.>"
~;",\

t

......... '"
-~
......:---. . ,.
r- :J'-""- ~QL:.--,""""',--_~
..u.

Glucose compared to CO2 and H 2 0 Is rich In energy.

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Energy Qeneratlon Is a mojor purpose of metabolism.
Other purposes:
1- synthesis of building blocks.
2- synthesis of macromolecule
3- degredatlon of blomolecules

Metabolism: is the sum of all chemical reactions in all living


organisms.
Can be divided Into 2 types:
1) catabolism: large molecules (energy yellding nutrients) are
degraded Into energy poor ent products like: CO2 , H 2 0.
2) anabolism: the small molecules like amino acids, sugars ,
fatty acids, nitrogenous bases are converted into large
molecules like proteins I polysaccharides, lipids and nucliec
acids.

Bioenergetics:deals with energy transformation In the cell from


one form to another.
We use chemical energy (n':ltrients) converted to:
1) concentration gradient: Is the presence of molecules or Ions in
different concentration across the membrane.
Example:
Na is more concentrated outside the cell than inside (high energy
state).
2) electrical gradient
3) mechanical gradient
4) heat and even light

Bioenergetics Involve chemical processes and quantitative


aspects of energy transformation. This energy transformation
follows the same rules that govern the nonliving systems.

Prediction of spontaneity or reactions:


Spontaneous means possible ( possible doesn't mean it is a fast
reaction or it will actually occur In the cell.)
Non· Spontaneous (not possible): cannot occur unless energy is
provide.

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Note:
Thermodynamics help to understand the spontaneity of the
reaction.

Which thermodynamic function can be used to determine the


posibility of a rxn??

The thermodynamic function that thought to be useful In


predicting the reaction possibility Is the enthalpy! heat content!
heat transformation.

Hot object can be a test tube, a cell ,an organism ,or an animal.
The heat can flow easily from a hot object to cold object.
This hot object will lose its energy! enthalpy to the surrounding.

Loss of heat is a Spontaneous process.


Heat can flow from the system to the surrounding or from the
surrounging to the system.

Thank you,
Your colleague,
Haya AI-Qudah

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