Antibodies: Structure and Function

Antibodies

• Glycoproteins (immunoglobulins or Ig) are produced in response to antigens
• Immunoglobulins recognize and bind to the antigens that caused their production.
• Antigen-binding proteins on the B-cell membrane are also known as antibodies
• Plasma cells secrete antigen-binding proteins or antibodies
• Membrane-bound antibodies give B-cell clones antigenic specificity
• The interaction of a membrane antibody with its antigen causes antigen-specific B-cell clone proliferation
• Humoral immunity is carried out by secreted antibodies in the blood
• Antibodies neutralize or eliminate antigens by seeking them out

Antibody Function and Structure

• Antibodies bind antigen
• Antibodies share structural features
• Antibodies participate in a limited number of effector functions
• Antibody responses to particular antigens are heterogeneous because antigens are complex and contain many different antigenic determinants
• The immune system responds to epitopes on the antigen, requiring recruitment of several B cell clones, resulting in monoclonal antibodies
• Monoclonal antibodies bind specifically to a single antigenic determinant
• Polyclonal and heterogeneous serum antibody response occurs in an immunized antigen
• Antibodies appear in blood serum and tissue fluids of vertebrate animal mucosal surfaces

Antibody Structure

• The amino-terminal variable (V) region, consisting of 100-110 amino acids, differs in each heavy and light chain of an immunoglobulin molecule
• Constant (C) regions exhibit limited variation defining two light-chain subtypes and five heavy-chain subclasses
• The amino terminal portions of heavy chains (y, δ, and α) corresponding to the V regions which bind to the antigen contain a proline- hinge region
• Effector functions are mediated by the other domains
• μ and ϵ heavy chains lack a hinge region, however, they contain an additional domain in the middle of the molecule
• Immunoglobulin molecules are Y-shaped, all having four polypeptide chains connected by disulfide bonds
• Two light (L) chains with approximately 220 amino acids each make up the structure with a mass of 25kDa each
• Two identical heavy (H) chains are apart of the structure, each consists of about 440 amino acids each and has a mass of about 50-70kDa
• Structurally distinct heavy chains exist for each immunoglobulin class or subclass

Light Chains

• Kappa (κ) and lambda (λ) are the two types of light chain, coded by different genes- although very similar structurally
• An individual antibody molecule contains two heavy chains and either two kappa chains or two lambda chains
• Kappa and lambda chains are never present together in the same antibody molecule
• Heavy chains are linked to each other and to the light chains by disulfide bridges
• Light chains may be either kappa or lambda
• Each polypeptide chain contains an amino terminal and a carboxyl terminal
• Light and heavy chains both contain constant and variable regions
• Antibodies of the same class have constant regions (CL and CH) with highly similar amino acid sequences
• Variable regions (VL and VH) have different sequences
• Antigen-binding sites are formed by the variable regions (VLVH) that have been folded together

Chains

• Four chains are arranged in a flexible Y shape with a hinge region, allowing a T shape.
• The crystallizable fragment (Fc) is the stalk of the Y and contains the site at which the antibody molecule binds to a cell
• Antigen-binding fragments (Fab) are at the top of the Y which bind to compatible epitopes called antigen determinant sites
• Fc fragments are composed only of constant regions, whereas the Fab fragments have both constant and variable regions
• Each heavy and light chain contains several homologous amino acid units of about 100-110; within each unit, called a domain, disulfide bonds form a loop of approximately 60 amino acids
• Interchain disulfide bonds also link the heavy and light chains

Light Chain Forms

• Kappa and lambda are two distinct forms of light chains which are distinguished amino acid sequence of the carboxyl portion of the chain
• Carboxyl-terminal portion of all kappa chains which are founded in human immunoglobulins are identical and termed the constant (CL) domain
• Lambda chains have four similar sequences defining the subtypes λ1, λ2, λ3 and λ4 with corresponding constant regions Cλ1, Cλ2, Cλ3 and Cλ4
• Hypervariable regions within the light-chain variable domain, also known as complimentarily-determining regions (CDRs) differ in amino acid sequence more frequently than the rest of the variable domain

Heavy Chains

• The NH2 terminal domain in the heavy chain has similar variability patterns to the Vκ and Vλ domains, named the VH domain, and contains four hypervariable regions
• Constant domains of the heavy chains are numbered CH1, CH2, CH3, and CH4 starting next to the variable domain
• The constant domains of the heavy chain form the constant (CH) region

Immunoglobulin Chain Domains

• Light chains contain a single variable domain (VL) and constant domain (CL)
• Heavy chains contain variable domain VH and have either three or four constant domains (CH1, CH2, CH3 and CH4)
• Immunoglobulin regions that fold together in three-dimensions (VH, VL) form the antigen-binding sites

Light-Chain

• Immunoglobulins have constant and variable regions revealed by light-chain sequences
• The amino-terminal half of the light-chain, varying by protein, contains 100-110 amino acids and is called the variable (V) region
• There are two main amino acid sequences in the carboxyl-terminal half of the molecule which is the constant (C) region.
• Kappa (κ) and lambda (λ) are the two light chain types, where a single antibody molecule only contains one type, either κ or λ and never both
• Light chains are classified subtype based on small differences in amino acid sequences.
• Mice light chains have 3 subtypes and humans have 4 subtypes

Heavy-Chain

• Heavy-chain sequences reveal five basic varieties
• The amino-terminal part of the heavy chain (100–110 amino acids) shows great sequence variation, like the light chain, and is the variable (V) region
• The remaining part has five basic sequence patterns corresponding to heavy-chain constant (C) regions (μ, δ, γ, ϵ, and α)
• Heavy chains are called isotypes with constant regions approximately 330 amino acids for δ, γ, and α, and 440 amino acids for μ and ϵ
• Heavy chains of a given antibody molecule determine the class (IgM[μ], IgG[γ], IgA[α], IgD[δ], IgE[ϵ]) and can have either κ or λ light chains
• A single antibody molecule consisting of H2L2 has two identical heavy chains and two identical light chains
• The α and γ heavy chains are further classified into subisotypes, determining the subclass of antibody molecules based on their minor differences in the amino acid sequences
• Humans have two α heavy chain subisotypes—α1 and α2—(and thus two subclasses, IgA1 and IgA2)
• Humans have four γ heavy chains subisotypes: γ1, γ2, γ3, and γ4 and therefore four subclasses, IgG1, IgG2, IgG3, and IgG4

Immunoglobulin Structure

• Primarily determined by the primary, secondary, tertiary, and quaternary organization of the protein
• Primary Structure: The amino acid sequence accounting for variable and constant regions of the heavy and light chains
• Secondary Structure: Formation by polypeptide chain folding in an antiparallel β pleated sheet
• The light and heavy chains have homologous units of about 110 amino acid residues, which contain an intrachain disulfide bond forming a loop of about 60 amino acids called a domain
• Light chains contain variable domain (VL) and constant domain (CH) and either three or four constant domains (CH1, CH2, CH3 and CH4), depending on class
• Immunoglobulin domains are folded into a characteristic compact structure which is the immunoglobulin fold
• The immunoglobulin fold contributes to the quaternary structure by facilitating noncovalent domain interactions across the ẞ sheets

Domain interactions

• Interactions form links between identical domains (e.g., CH2/CH2, CH3/CH3, and CH4/CH4) and between nonidentical domains (e.g., VH/VL and CH1/CL)
• Loops contain the VH and VL sequences which contain variable amino acids and constitute the antigen-binding site of the molecule
• The three (VL and VH) loops of each variable domain show considerable variation and high variability which constitute the antigen-binding site of the molecule
• The structure of the epitope is complementary to the antigen binding site, therefore hypervariable regions are usually called complementarity-determining regions (CDRs)
• A wide range of specificities is exhibited by antibodies due to variations in the length and amino acid sequence of the six CDRs in each Fab fragment

CDR Residues

• The CDRs are the antigen-binding regions of antibodies and have more residues in the heavy-chain that contact the antigen in the light-chains
• VH contributes more to antigen binding, however in some antibody-antigen reactions, the light chain makes the more important contribution
• Antigen binding induces conformational changes in the antibody, antigen or both
• Neuraminidase and anti-neuraminidase binding is accompanied by side chain orientation change of both the epitope and the antigen-binding site

Immunoglobulin Domains

• Constant region domains serve a role in biological functions and determined by amino acid sequence of each domain
• The CH1 and CL domains extend the Fab arms of the antibody molecule to facilitate interaction with antigen and have increased rotation
• Constant-region domains help to hold the VH and VL domains together by the interchain disulfide bonds
• CH1 and CL contribute to antibody diversity by allowing random association between VH and VL domains
• Random rearrangements of immunoglobulin genes generate unique VH and VL sequences for the heavy and light chains from each B lymphocyte
• VH and VL sequences generate a unique antigen binding site
• Increased stable VH and VL interactions from the presence of CH1 and CL domains contributes to the overall diversity of antibody molecules

Hinge Region

• The γ, δ, and α heavy chains contain a peptide sequence between the CH1 and CH2 domains that has no homology with the other domains
• Rich in proline residues and is flexible, giving IgG, IgD, and IgA segmental flexibility which allows the two Fab arms to assume various angles

Key Hinge Characteristics

• The two prominent amino acids in the hinge region are proline and cysteine
• Cysteine residues form interchain disulfide bonds which holds together the two heavy chains where the number can vary between different classes of antibodies
• The μ and ϵ chains lack a hinge region, but have an additional 110 amino acid domain (CH2/CH2) that has hingelike features
• The heavy chains in IgA, IgD, and IgG contain 3 constant-region domains and a hinge region, while the heavy chains in IgE and IgM contain four constant region domains and no hinge region
• CH3/CH3 designates the carboxyl-terminal domain in IgA, IgD, and IgG while CH4/CH4 designates the carboxyl-terminal domain in IgE and IgM

Antibody Subclasses

• Fine classes of antibody and their subclasses are expressed as secreted immunoglobulin (sIg) or as membrane-bound immunoglobulin (mIg)
• B cells express different classes of mIg through different developmental stages where immature B cells called pre-B cells only Express mIgM
• Mature B cells coexpress mIgD and IgM on the surface, before being activated by an antigen
• A memory B cell expresses mIgM, mIgG, mIgA, or mIgE and the antigenic specificity of all the membrane antibody molecules are identical on a single cell to bind the same epitope.

Immunoglobin Classes

• Heavy chain constant region amino acid sequences determine immunoglobulin classes in humans where there are five classes or isotypes.
• These classes or isotypes are designated by their heavy-chain:
  • IgG: gamma (γ) heavy chain
  • IgA: alpha (α) heavy chain -IgM :mu (μ) heavy chain -IgE: epsilon (ε) heavy chain -IgD: delta (δ) heavy chain

Ig Hypervariable Regions

• The terms ‘hyper-variable region’ and ‘complementarity-determining region’ means the same thing
• The antigen-binding site of the antibody is complementary in shape to the antigenic epitope- ensuring the shape fits
• The hyper-variable regions of an antibody are what determine whether it is complementary to an antigenic epitope, and so they are the complimentarity-determining regions (CDRs)

Biologic Functions

• Amino acid sequence in the heavy chain constant region determines classes of immunoglobulins and confers class-specific structural and functional properties
• The structure and function are shown in these human heavy chain classes or isotypes:
  • IgG: gamma (γ) heavy chain
  • IgA: alpha (α) heavy chain
  • IgM :mu (μ) heavy chain
  • IgE: epsilon (ε) heavy chain
  • IgD: delta (δ) heavy chain

Immunoglobin G

• IgG is the most abundant class in serum that constitutes approximately 80% of the total immunoglobulin of the serum
• Four human IgG subclasses are numbered average serum concentrations: IgG1, IgG2, IgG3, and IgG4 and are distinguished by differences in γ-chain sequences
• DNA sequences 90%–95% homologous different germ-line CH genes, encode the amino acid sequences which distinguish the subclasses of IgG
• The hinge region size as well as the position and number of interchain disulfide bonds between heavy chains are structural characteristics distinguishing IgG
  • Approximately 65% of total serum IgG is IgG1, and 23% isIgG2
• Subtle amino acid differences affect the biological activity of the IgG subclasses
• IgG1, IgG3, and IgG4 cross the placenta and protect the developing fetus
• IgG3 is the most effective complement activator and is followed by IgG1 while IgG2 shows less efficiency
• IgG4 is unable to activate complement.
• IgG1 and IgG3 bind with high affinity to Fc receptors on phagocytic cells; thus mediate opsonization
• IgG4 has an intermediate affinity for Fc receptors, and IgG2 has extremely low affinity
• IgG2 develops in response to antitoxins
• IgG4 antibodies function as skin sensitizing immunoglobulins -Anti-Rh antibodies are of the IgG1 or IgG3 subclass.

Immunoglobin M

• Accounts for 5%–10% of the total serum immunoglobulin, with an average concentration of 1.5 mg/ml
• Monomeric IgM, with a molecular weight of 180,000, is expressed as membrane-bound antibody on B cells and is secreted by plasma cells as a pentamer
• Five monomer units are held together by disulfide bonds which link their carboxyl-terminal heavy chain domains
• Five monomer subunits that are arranged with their Fc regions in the pentamer center while the ten antigen-binding sites are on the periphery
• Each pentamer has an additional Fc-linked polypeptide, known as J (joining) chain which is disulfide-bonded to the carboxyl- terminal cysteine residue of two of the ten μ chains
• J chain is required for the polymerization of the monomers to form pentameric IgM.
• J chain is added just before secretion of the pentamer.

IgM Biological activity

• Produced in a primary response to an antigen
• It is the first immunoglobulin to be synthesized by the neonate
• Produced during B-cell maturation
• Expressed as a membrane-bound antibody on B cells Serum IgM has high valency due to its pentameric structure and 10 antigen-binding sites
• Pentameric IgM is more efficient than isotypes in binding antigens with many repeating epitopes due to valency,
• Less IgM is required than IgG to neutralize viral infectivity
• IgM is more efficient at activating complement
• IgM, because of it's size is constrained to blood and cannot cross the placenta

Immunoglobin A

• Constitutes small amount of total serum immunoglobulin from 10%-15%
• Predominant immunoglobulin class in external secretions(breast milk, saliva, tears, mucus)
• Secreted in monomers, and polymeric forms(dimers, trimers) are sometimes encountered. and the polymeric forms containing a J-chain polypeptide
• Secretory IgA, found in external secretions, and consists of a dimer or tetramer, J-chain polypeptide and a polypeptide chain known as secretory component

IgA Function

• J-chain IgA polypeptide id identical to IgM performing a similar function in the polymerization serum with IgA
• Mucous membranes of the epithelial cells produce a Secretory component(70,000-MW polypeptide)
• Consists of five immunoglobulin-like domains that bind to the Fc region domains of the IgA dimer
• This interaction is stabilized by a disulfide bond between the fifth domain of the secretory component and one of the chains of the dimeric IgA.
• IgA produces more than any other the other immunoglobulin chain and are along mucous membrane surfaces where daily a human secretes from 5 g to 15 g of secretory IgA into mucous secretions

IgA Biologic Activities

• Serves vital function at mucous membrane surfaces where most pathogenic organisms enter
• Cross-links large antigens with multiple epitopes because they are polymeric
• Attaches to antigens of viral surfaces prevent their pathogen from attaching to mucosal cells inhibiting infections.
• Provides important defense against pathogens, also defends polio, seasonal viruses and reovirus
• Breastmilk of a newborn helps protect during the first few months while their immunity system is still developing

Immunoglobin E

• Was identified in serum despite average concentration being very low .3ug/ml because it shows potent biologics action
• Is a IgE that has hypersensitivitiy to and controls response to hay fever, asthma, hives, and anaphylactic shock at a moments notice

IgE Activities

• IgE molecules bind to Fc receptors of membranes of blood basophils and tissue mast cells
• Induces cross-linkage with receptor-bound IgE by antigen-allergin, to prompt cells to react and release pharmacologically active mediators which induces allergic reactions
• Localized mast-cell process induced by IgE may release mediators that facilitate antiparasitic cell defense.

Immunoglobin D

• Makes up a very small percent in Serum concentration for only .2% at 30ug/ml
• It teams with Igm as major contributor molecule expressed by mature B- Cells
• Displays no biological effector function.

Ab Effector Functions

• While antibodies respond to antigen they also respond with cellular response to kill pathogens These responses (effector functions) help cause the pathogens to be removed and killed through collaborative interactions that are specific Antibodies protect against infectious agents and work with the immune system They are:

1. Neutralization
2. Agglutination
3. Opsonization
4. Activation of complement
5. Antibody-dependent cell mediated cytotoxicity

• One class of antibody neutralization helps with variety of pathogens

Pathogens Nuetralized

• This is very important for tetanus, diphteria and botulism diseases
• Viral infection also cause antibody to stop binding to the cell preventing infection
• Antibody especially IgA can prevent entry into the body
• Anti-body also prevents the anti-body from replicating with measles and influenza
• It helps prevent adherence to cells and prevents bacterial metabolism.

Ab Production

• Because it is a result of their multivalent structure they bind effectively and create processes of microb and antibody
• Agglutination limits spread of pathogens, and makes it more to be phagocytosed and killed

Opsonization

• Opsonization is antibody promoted phagocytosis of antigens which is important for defenders
• Fc works to bind contant region of Ig to macrophage and neutriphils
• Antibody triggers signal -transduction membrane in results phagocytosis of antigen

Compelment Activation

• IGM assists humans and most Igg subclasses in complement system
• IGM C3b binds to cell and cells and has receptors for C3b
• C3b leads to macrophages and cell to be attacked
• Binding of Antigen Antibody and collaboration between antibodies helps to help destroy compnent

AB Mediated Cytotoxicity

• Connecting or binding with another protein cell-mediated functions kills the cell
• AB becomes a newly effective receptor to help recognize and kill the cell
• Neutriphils monocytes, killer helps kill IGG as ADCC but ADCC helps attach and kill, but is not effective at engulfing cell
• If AB Binds to help the cell ADCC helps killer cells bind to the cell

Transcytosis Epithellial Layers

• Helping transfer AB to the mucosal layers helps with transytosis
• Transfer is important in this region which IGA is very helpful too
• In the same way transfer helps fetus for development over 3 months.

B-Cell AB Transfer

• Iso-types have short cy toplasmic tails
• Tail Is short to function with intracellular molecule such as tail short (GPR)
• This short tail does not work with BCells it has a transmembrane that is effective

AB Isotype Switch

• This is a cellular mechanism that switch the production of AB type
• Process swaps AB chain while the original heavy chain remains
• This original heavy chain the original and can attach while in affector cells

Chain switch

• AB is switched after mutation which generate different class and is similar to the original recomb
• Naíve in the bone produce chain segments when activated cell molecules proliferate

Learn about antibodies, glycoproteins produced in response to antigens. Explore their structure, function, and how they neutralize or eliminate antigens. Discover how antibodies participate in a limited number of effector functions.