Rebecca martin/sandbox2

Introduction to IgAIntroduction to IgA

spinqualitylabelsall models Forms of IgA Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The most extensive surface in contact with the external environment is not our skin, but the epithelial lining of our gastrointestinal, respiratory, and urogenital tracts . As a first line of defense in maintainance the integrity our mucosa, the immune system manufatures and secretes dimeric IgA to neutralize pathogenic organisms and exclude the entry of commensals at the mucosal border . In the serum, IgA functions as a second line of defense against pathogens that may breech the epithelial boundary . The body produces more IgA than any other antibody isotype . In fact, IgA is the most abundant antibody in the body, further illustrating IgA's critical role in immunity .

Unlike other antibody isotypes, IgA exists in mutiple oligomeric states . The most common of which are the monomeric, dimeric, and secretory forms . At least two isotypes exist, termed IgA1 and IgA2. IgA2 can further be categorized into 2 allotypes: IgA2 m(1) and IgA2 m(2). The receptors for IgA include the Fcα Receptor (FcαRI; CD89) and the polyimmunologlobulin receptor (pIgRI). When binding to FcαRI results in the dimerization, the consequent signaling results in effector functions, including respiratory burstmucosal surface, an approximately equal ratio of secretory IgA1 (sIgA1) to secretory IgA2 (sIgA2) reside at the mucosal surface, with the exception of the colon, where the majority is sIgA2 . In the serum, about 90% of the IgA is monomeric IgA1 , phaocytosis, and eosinophil degranulation. Binding to the pIgR results in transoocytosis and IgA secretion . Exploring IgA's structure and protein interactions illuminates the unique and critical function IgA plays in humoral immunity.

Antibody Structure and the Immunoglobulin DomainAntibody Structure and the Immunoglobulin Domain

Overall Structure

An antibody is a tetramer of and . In other words, the antibody is a homodimer of 2 heterodimers. Each is comprised on one light chain and one heavy chain. Heavy and light chains are held together with disulfide bonds and noncovalent interactions.

Fab and Fc fragments

Another common way of describing antibody structure is in terms of its Fab and Fc fragments. Each light chains are composed of 2 immunoglobulin domains: one variable domain</scene> and one constant domain. Heavy chains composed of 4 Ig domains: one V-type and 3 C-type, named CH1 - CH3. A linking hinge region separates the CH2 and CH3 domains. Proteolytic cleavage at the hinge region by the protease papain, or a similar protease, yields 2 Fab fragments and 1 Fc fragment. Each contains 2 variable domains, one from the heavy chain and one from the light chain, and 2 constant domains one from the light chain and the Ch1 domain from the heavy chain. The Fc fragment __________ contains 4 constant domains: the Ch2 and Ch3 domains from each of the heavy chains. Since the variable portions determine antigen specificity, the Fab fragments are generally thought of as the antigen-binding portion. The Fc fragment is important in binding various receptors, many of which are isotype specific and are named after the isotype of the ligand, i.e. FcαR binds the Fc portion of IgA.

Immunoglobulin domains

The antibody is a member of the immunoglobulin superfamily of proteins (ref Att). Each chain can be further broken down into immunoglobulin domains: 2 in the light chain and 4 in the heavy chain, for a total of 12 in the entire antibody. Each immunoglobulin domain contains a primary amino acid sequence of approximately 70 – 100 residues long. Secondary structure is a characteristic beta sandwich with a variable number of beta strands, depending on the unit type. These strands display Greek key connectivity (web other) and form 2 beta sheets that fold over each other. An intra-domain disulfide bond stabilizes the tertiary structure.

Nine antiparrallel beta strands comprise variable or V-regions. Loop sequences of varying length connect the strands. The 9 strands form 2 beta sheets, one with 4 (ABED-prosite) strands and the other with 3 (CFG prosite). The remaining 2 strands (C’ and C”) lie in between the 2 sheets. A disulfide bride stabilizes the 2 sandwich halves. Hydrophobic residues face the interior of the sheet, providing stability, while hydrophillic residues face outward and interact with the local environment. The extra loops in the V-region are critical for epitope specificity, and are consequently known as the compliment determining regions, here shown on the Fab molecule .

C-type domains lack the C' and C beta strands. The sheets are ABED and CFG. Consequently, the sandich is more tightly packed. In the antibody, the constant domains determine the isotype: IgA, IgD, IgM, IgG, or IgE.

• • Related structures

Proteins containing the classic immunoglobulin-like domain are found predominantly in the immune system. In fact, the antibody's closest related structires are those that recognize antigen: MHC and TCRs.

The V-type domain is found in a wider variety of proteins, including the Ig-binding molecules, such as the pIgR and the FcalphaR.

Viral hemagluttinin is yet another example.

IgA1 and IgA2IgA1 and IgA2

Two isoforms of monomeric IgA exist: IgA1 and IgA2. While IgA2 is present in most mammals, IgA1 is only present in higher apes 15111057. While the ratios of secretory IgA1 and IgA2 are approximately 1:1 in most mucosal locations, IgA2 predominates in the colon 19109255. As stated earlier, the form of IgA1 in the serum is monomeric IgA1.

Major structural differences between the IgA1 and IgA2 monomers can be seen in the hinge region, and glycosylated residues,

IgA2 can further be categorized into two or more allotypes. The hinge region differs significantly between the two isoforms. The hinge region of IgA1 is comprised of 23 residues (PVPSTPPTPSPSTPPTPSPSCCH) and 5 O-glycosylation sites, while IgA2’s hinge region is comprised of 10 residues (PVPPPPPCCH) and no sites of glycosylation. Both hinge regions are located at Cys220 on the Ch1 chain and end at Ch2’s Pro244; however, the naming system is misleading, as it follows IgA1 and is therefore misleading. In fact, the distance from the the center of the 2 Fab fragments in IgA1 ia 16.9nm versus 8.2 nm in IgA2. So, while IgA1 remains extended, IgA2 is more compact. The greater number of residues in the IgA1 hinge region corresponds to a greater antigenic reach 15111057.

These data must be taken into account with other hinge region characteristics. IgA1’s hinge region contains 5 sites of O-glycosylation, while IgA2’s hinge region contains none. In addition, IgA1’s hinge region contains 10 Pro residues, while IgA2’s region contains 6. In comparison, IgG’s hinge region contains No glycine residues reside in the hinge regions of either IgA1 or IgA2. The presence of prolines, the absence of glycine and the presence of glycosylated residues in IgA1 all amount to increased hinge rigidity in comparison to IgG1.

In the harsh mucosal environment, glycosylated residues protect the protein from proteases. Both IgA1 and IgA2 display N-glycosylated residues. IgA1 has 3, at N263 on beta strand B on the Ch2 chain and on the J tail at N459. In IgA2, additional sites of N-glycosylation include Asn166 on the beta strand G of Ch1 and Asn337 of beta strand G on Ch2. Some alloforms of IgA2 are also N-glycosylated at Asn211 on Ch2. 15111057 An increased need for protection against proteolytic cleavage at the hinge region accounts for the presence of O-glycosylation in IgA1’s hinge region, particularly cleavage by bacterial metalloproteases. The glycosylation residues provide increased steric hindrance, and creating difficulty in fitting the peptide in the protease’s active site. In comparison to IgG, which is only 2.9% (w/w) glycosylated, IgA1 is 9.5% (w/w) and IgA2 is 11% (w/w) glycosylated. Overall, IgA1 is more susceptable to proteases than IgA2.

The two structures also differ in the locations of their disulfide bonds 15111057 . In IgA1, a disulfide bond exists between the heavy chain Cys220 and light chain Cys196. This disulfide bond is absent in the main form of IgA2. The heavy and light chain assiciate through noncovalent interactions. So, while IgA1 may be more susceptable to proteases, IgA2 is more susceptable to denaturing conditions.

Compare and contrast moduleCompare and contrast module

spinqualitylabelsall models monomeric IgA1 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Hinge length

Hinge glycosylation

Hinge Prolines

N-glycosylated residues

Disulfide bonds

spinqualitylabelsall models monomeric IgA2 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Hinge length

Hinge glycosylation

Hinge Prolines

N-glycosylated residues

Disulfide bonds

Dimeric IgADimeric IgA

spinqualitylabelsall models dimeric Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

J chain, 10064707 18 kDa, Ig-related. 137 residues, 1 N-linked oligo Binds @ Cterm Ch3 domain 18178841 covalently attached via disulfide bridge w Cys 471 10064707 N linked glycosylation site 15111057 T shape: 180 degrees inter-fab angle vs iGG- Y shaped 120 degrees 15111057; 10064707 Hinge region: less flexible than IgG (Pro, O glycosylation, no glycine = swivel points) 15111057 (IgG- 17-23 residue hinge, glycines, few Pro, no glycosylation) 15111057 2 FcalphaRI binding sites, one per heavy chain, at each Ch2-Ch3 interface – both domains contribute 2:1 stoichiometry 15111057 Dimer/ polymer: intracellular, J chain + inter IgA disulfide bridge 10064707 Polymer rare 2/2 steric hindrance of T-shaped fab 18178841 Cys 471 disulfide bridged w either cys14 or cys 68 in j chain 18178841 IgA1: near planar, Fc End to end w/o overlap, Fc-Fc length ~14.4nm 18178841 J chain doesn’t act as spacer, lies in crevice of bent Fc regions 18178841 J chain may then have access to SC part of pIg, noncovalent  (hypothesized unfolding and wrapping?) translocation 18178841

Secretory ComponentSecretory Component

spinqualitylabelsall models secretory component. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Polymeric immunoglobulin receptor found @ basolateral side of CM 10064707 IgA binds  luminal side. 10064707 EC region cleaved  secretion + disulfide forms 10064707 Funct: helps prevent entry of pathogens/ gut flora into mucosa 10064707 SC steric hindrance  pathogens cannot bind to mucosal surface 12768205 5 Ig-like domains = D1-5; 7 or less glycan chains 17428798 Function binding to IgA = protection against proteolytic degradation 17428798 Free = antibacterial – various pathogenic bacteria, H pylori, C dif; oligosacc help 17428798 = 1st 585 residues of pIgR 17428798 Structure = D1 (Hu, Rb), D2 (Rb,Ms), D3 (Ms) ~ Ig V-type superfam; 7 beta strands A-G and C’ and C’’ 17428798 Glycosylation + linking regions btwn domains- longer = less likely to be crystallized 17428798 Compact arrangement 17428798 Length: D1-3 ~12nm; D4-5 ~10nm 17428798 Free SC – compact structure, folds on itself in J shape @ D3  D4 domains (long 10 aa linker), D1 remains accessible 17428798 Susceptable to proteases Arg336/Ser337 17428798 D2-D3 linker is short, facilitating folding 17428798 Cterm linker to TM domains of pIg – no specific fold, moves freely, facilitates release 2/2 proteolytic cleavage 17428798 7 glycan residues- mostly on one side, facilitates binding, away from protein surface (likely contributes to sIgA binding to m/o surface?) 17428798 One-side distribution allows free access 3 CDRs at D1 and Cys502 at D5 17428798 Glycan residues do not impact binding aff to Iga 17428798 Oligos important w resistance to proteases and anchoring @ mucosal surface 17428798 Fc iga = more susc to intestinal proteases, precisely region pIg binds to and SC remains in assoc w Fc portion, w/o affecting function or motility of Fab or hinge region 17428798 Assoc  “zipper effect”  D1 assoc w a constant chain of IgA  J chain  disulfide forms Ch2 C311 and D5 C502 17428798 Binding to Fc  reduce flexibility @ hinge and btwn 2 Fc regions  less lilkely to be in correct conformation for cleavage to occur 19079336

Secretory IgASecretory IgA

spinqualitylabelsall models secretory Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

spinqualitylabelsall models secretory IgA1 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Secretory: polymeric (mostly dimeric (can as more) 10064707 Cys 311-Cys SC 10064707 SC interacts w DE and FG loops 12768205 SIgA  steric hindrance, no binding to mucosal surface 12768205 SIgA cannot activate Fcalpha R, FG loops = major binding site for Fc 12768205 C311 only 10 angstroms away 12768205  no binding to Fcalpha/ no activation w/o integrin 12768205 In addition cyt okines modulate activity, thought to occur through modulation of Fcalpha’s surface density (requires dimerization to signal) 12768205 Unfolds upon binding, no change in dIgA structure 19079336 SC delays cleavage at Fc and hinge region, decreased access 2/2 fab and binding to cell surface R (bacterial proteases are alrge) 19079336

Insights into FunctionInsights into Function

Ag binding and effector functions (not conf change: Fc – Fc interaction or assoc mutiple ag-ab complexes 10064707 Fc interacts w Fcalpha at edge not face 19079336 Glycosylation = more resistant to proteolytic attack + IgA wide separation (protects hinge region) 10064707 Asn263 O-glycosylation = protection 10064707 Fc iga = more susc to intestinal proteases, precisely region pIg binds to and SC remains in assoc w = optimized molecule for function @ harsh mucosal env. 17428798 Fcalpha theoretically binds x4 to dIgA (occurs independent of Ag binding), but dIgA only permits 2 binding sites, steric hindrance. SC blocks 2 sites- so 1:1 19079336  required multiple binding for Fcalpha/ clustering Limit inflammation @ mucosal surface (me)

Implications in Science and MedicineImplications in Science and Medicine

Proposed mechanism for IgA nephropathy Sx: Pathology: Dz caused by polymeric IgA1 deposited @ kidney glomeruli 18178841 Lack of the nepropathy in ppl w IgA myeloma w/o nephropathy  abnormal IgA Propose disturbance in hinge region/ absence of fab (Steric hindrance of T-shaped fab regions  polymers rare) Decreased O-glycosylation has been proposed as a mechanism- may destabilize hinge region, allow IgA to self associate or allow cleavage of hinge region by bacterial proteases Conclusion: near-planar characteristic lends IgA1 to pathology 2/2 formation multimers following disruption of fab fragments from their natural rigid form

Limitations of the Current StudiesLimitations of the Current Studies

10064707; 15111057 xray and neutron scattering analysis + analytical ultracentrifugation and analyzed w constrained modeling 2/2 high carb and flex = difficult to crystalize 18178841

Questions for the FutureQuestions for the Future

Because of the limitating resolution of these models, many details concerning the binding residues and residue interactions are left unknown. Crystallographic structure will yield further insights into the structure of IgA, the interactions between IgA and other molecules, and ....

SC aa interact w J chain? CDR-like motifs @ D1 binds ? @ IgA; Does SC open upon binding?; stoichiometry of binding? Locations of oligos on SC? Differences in binding IgA1 vs IgA2 17428798 Binding motifs SC and IgA1 18178841 Structure of IgA involved in IgA nephropathy 18178841

LinksLinks

IgAIgA

• Fab and Fc Fragments

Refined crystal structure of the galactan-binding immunoglobulin fab j539 at 1.95-angstroms resolution 2fbj

Phosphocholine binding immunoglobulin fab mc/pc603. an x-ray diffraction study at 2.7 angstroms 1mcp

Phosphocholine binding immunoglobulin fab mc/pc603. an x-ray diffraction study at 3.1 angstroms 2mcp

Crystal structure of human FcaRI bound to IgA1-Fc 1ow0

Refined crystal structure of a recombinant immunoglobulin domain and a complementarity-determining region 1-grafted mutant 2imm and2imn

Crystal structure of a Staphylococcus aureus protein (SSL7) in complex with Fc of human IgA1 2qej

• Monomeric

Model of human IgA1 determined by solution scattering, curve-fitting, and homology modeling 1iga

Model of human IgA2 determined by solution scattering, curve fitting and homology modelling 1r70

• Dimeric and Secretory

Solution structure of human dimeric immunoglobulin A 2qtj

Solution structure of human secretory IgA1 3chn

Solution Structure of Human SIgA2 3cm9

Solution structure of human secretory component 2ocw

Related MoleculesRelated Molecules

• non-IgA antibody isotypes

IgM: Solution structure of human Immunoglobulin M 2rcj

IgG:

IgD:

IgE:

• Other C-type immunoglobulin examples

MHC: Crystal Structure of monomeric human beta-2-microglobulin 1lds

TCR: Crystal Structure of the G17E/A52V/S54N/Q72H/E80V/L81S/T87S/G96V variant of the murine T cell receptor V beta 8.2 domain 2apv

• V-type immunoglobulin examples

Crystal Structure of a Ligand-Binding Domain of the Human Polymeric Ig Receptor, pIgR 1xed

Crystal structure of human FcaRI 10vz