Rebecca Martin/Sandbox1: Difference between revisions

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 <ref name="seven">PMID:17428798</ref>. As a first line of defense in maintainance the integrity our mucosa, the immune system manufatures and secretes dimeric IgA to neutralize pathogenic organisms <ref name="five">PMID:15111057</ref> and exclude the entry of commensals at the mucosal border <ref name="nineseven">PMID:19079336</ref>. In the serum, IgA functions as a second line of defense against pathogens that may breech the epithelial boundary <ref name="five" />. The body produces more IgA than any other antibody isotype  <ref name="nineseven"/>. In fact, IgA is the most abundant antibody in the body, further illustrating IgA's critical role in immunity <ref name="ten">PMID:10064707</ref>.   

The receptors for IgA include the Fcα Receptor (FcαRI; CD89) and the polyimmunologlobulin receptor (pIgR). When binding to FcαRI results in the dimerization, the consequent signaling results in effector functions, including respiratory burst, mucosal surface, phaocytosis, and eosinophil degranulation. Binding to the pIgR results in transoocytosis and IgA secretion <ref name="five" />. Unlike other antibody isotypes, IgA exists in mutiple oligomeric states <ref name="nineseven" />. The most common of which are the monomeric, dimeric, and secretory forms <ref name="ten" />, adding to the complexity of structural functions for IgA. Exploring IgA's structure and protein interactions illuminates the unique and critical function IgA plays in humoral immunity.

::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 sIgA2(nov 22 2007) <ref name="CFG">(nov 22 2007). "Superfamily: immunoglobulin." SCOP, from http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.c.b.b.html.</ref>. 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 <scene name='Rebecca_Martin/Sandbox1/Cdr_360_view/2'>Fab molecule</scene>.

::C-type domains lack the C' and C<nowiki>''</nowiki> beta strands <ref name="att" />. The sheets are ABED and CFG. Consequently, the sandwich is more tightly packed. In the antibody, the constant domains determine the isotype: IgA, IgD, IgM, IgG, or IgE.

::Proteins containing the classic immunoglobulin-like domain are found predominantly in the immune system <ref name="att" />. 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 <ref name="att" />. Viral hemagluttinin is yet another example.

:The hinge region differs significantly between the two IgA isoforms <ref name="five"/>. 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'''.  

:In the harsh mucosal environment, glycosylated residues protect the protein from proteases <ref name="five"/>. 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. 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 <ref name="five"/>. 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. Instead a disulfide bond links the 2 light chains at their C termini. The heavy and light chain associate through noncovalent interactions. So, while IgA1 may be more susceptable to proteases, IgA2 is more susceptable to denaturing conditions.  

:The unique characteristics of IgA1 and IgA2 explain the antibodys' overall T-shape <ref name="five"/>. IgA distinctly lacks the classic "Y-shape" antibody structure. IgA's increased hinge rigidity and a longer hinge region result in IgA1's predominately T-shape, in comparison to IgG's Y-shape. While the structure of IgA2 is more compact, the combination of an inter-light chain disulfide bond, a short hinge region, and proline residues with the hinge provide steric forces compatable with a T-shape. Of note, the T-shaped IgA2, with its interchain disulfide bond, resembles the structure of an IgG lacking the disulfide bonds between the heavy and light chains, which suggests the possibility of an evolutionary relationship between the two. The presence of IgA2 in lower mammals in contrast to IgA1 also supports this hypothesis.   

<scene name='Rebecca_Martin/Sandbox1/Iga1_hinge_ser_thr/1'>Potential Sites of O-linked Glycosylation (5 residues per hinge glycosylated)</scene> Protect from proteases and increase hinge rigidity. Note the extended hinge region of 23 amino acids, extending IgA1's antgenic reach.

:When IgA forms dimers, the Fc regions align end to end without overlap <ref name="eight"/>. The J chain lies within a fold in the bent Fc region. This conformation may allow the J chain access to the Secretory Component of the pIgR, which allows translocation across the mucosal epithelia to the luminal surface. Of note, in the image the J chains the J chains are extending from the dimer, which does not match with the described interaction of the J chain with the Fc portions of the anitbody (see Limitations of the Current Studies).

 

:The secretory compenent is the first 585 residues of the pIgR <ref name="seven"/>. The C terminal end of the secretory component is linked to the pIgR, but maintains no specific fold. The ability for the secretory to move freely facilitates its proteolytic cleavage and the secretion of sIgA. Structurally, the secretory component is comprised of 5 V-type immunoglobulin-like domains (D1-5) with 5-7 glycan chains, which increase the chains resistance to proteases. These glycosylation sites are located on one side of the protein and do not interfere with IgA binding. A long (10 amino acids) linker region exists between D3 and D4, so the D4 and D5 regions fold in on D2 and D3 in a compact J-shape. D1-3 are 12nm in length, while D4-5 are 10 nm long. Thus, D1 remains accessable. The one-sided glycans allow free access of D1's CDR regions and the Cys 502 at D5 to interact with IgA. It is thought that when D1 interacts with IgA's Fc region and the J chain,  allowing the secretory component to unfold and disulfide formation between D5 C502 and IgA's Ch2 C311. While SC unfolds upon IgA binding, this binding imparts no change on the structure of IgA <ref name="nineseven"/>.  

'''Limiting Effector Responses through Decreased FcalphaR Binding'''

<applet load='1ow0' size='300' frame='true' align='right' caption='Fc portion of IgA bound to FcalphaR' />

:The FcalphaR binding sites are located one per heavy chain at each Ch2-Ch3 interface. Both both domains contribute one binding site. So, the stoichiometry between monomeric IgA and the FcalphaR is <scene name='Rebecca_Martin/Sandbox1/Fc/3'>2:1</scene> <ref name="five"/>. The Fc portion is shown in red, and the receptor is in blue. Dimerization would increase this stoichiometry 4:1; however, 2 of the binding sites will be <scene name='Rebecca_Martin/Sandbox1/Siga1_sites_covered/1'>covered by the secretory component</scene>. Because of <scene name='Rebecca_Martin/Sandbox1/Fc/4'>orientation</scene> constraints, only 1 of the 2 remaining binding sites will be available to bind receptor. Therefore, physiologic stoichiometry is 1:1.  

:The binding of IgA to the Fcalpha receptor does not elicit a structural change in the antibody <ref name="two"/>. Effector function is elicited when multiple receptors bind and resultant clustering triggers signaling events. The 1:1 stoichiometry greatly limits Fcalpha clustering and consequent effector functions by effectively '''limiting the concentration of available antibody binding sites''' in the local environment, favoring neutralization in the absence of cytotoxic and inflammatory responses upon antigen recognition. Additional modulation occurs through internal signaling events. For example, cytokines strigger changes in cytoskeletal arrangments that result in clustering of the FcalphaR at the cell surface.  In effect, the interaction of the '''secretory component limits the effector and inflammatory responses''' upon antigen binding without limiting the ability of the antibody to neutralize pathogens or exclude commensals from breeching the mucosal barrier.  

:While both IgA1 and IgA2 are able to bind polysaccharide, IgA1 preferentially binds protein antigen, while IgA2 preferentially binds lipopolysaccharide lipid A <ref name="nineten"/>. This difference in structure can be explained, at least in part, by structural differences. The bindng of the secretory component to IgA1 results in a planar antibody with a wide, rigid antigenic reach. In contrast, secretory component binding to IgA2 results in a compact nonplanar form.

:Since <scene name='Rebecca_Martin/Sandbox1/Siga1def/1'>secretory IgA1</scene> is planar and more flexible, this might lend to '''antigen binding''' on proteins, which are larger and more variable <ref name="nineten"/>. Flexibility allows IgA1 access to a more diverse array of orientations. Likewise the more compact, nonplanar <scene name='Rebecca_Martin/Sandbox1/Siga1/1'>secretory IgA2</scene> might preferentially bind repeating patterns on fixed surfaces, like bacteria coating intestinal mucosa. It is intersting to note that IgA2 tends to induce signaling more slowly than IgA1 upon bindig FcalphaR. So, differences in isoform structure correspond to different antigen specificities and consequent differences in the roles each isoform plays in elciting mucosal immune responses.  

:The secretory component interacts with either dimeric IgA1 or IgA2 to form a '''functional unit''', structurally adapted to the harsh mucosal environment and to control potentially pathogenic mucosal flora primarily through neutralization. Inflammation is controlled by limiting the available binding sites on the Fc portion of IgA, effectively preventing Fcalpha clustering through a 1:1 stoichiometric binding. Differences in structure and resulting function allows the two isoforms fill unique niches in mucosal immune responses, suggesting selective advantages for each. Whereas IgA1 specializes in protein detection, IgA2 tends to bind LPS and polysaccharide antigen. So, structure arms the IgA secretory unit with specific advantages suited for its environmental, maintains balance between inflamation and mucosal barrier protection by limiting effector responses, and imparts unique functional roles to IgA isoforms. Together, structure and function determine the immune niches filled by IgA1 and IgA2.

== Implications in Science and Medicine ==

[[Image:IgA_IFA.jpg|thumb|Immunofluorescence detecting IgA in IgA glomerulonephritis. From http://www.unckidneycenter.org/images/IgA_IFA.jpg.]]

:IgA nephropathy is the most prebvalent cause of chronic glomerulonephritis in the world and is caused by polymeric IgA1 deposited @ kidney glomeruli <ref name="eight"/>. Notably, 90% of serum IgA is IgA1, mostly in the monomeric form.The observation that individuals with IgA myeloma [http://en.wikipedia.org/wiki/Multiple_myeloma] lack nephropathy suggests an abnormality in IgA structure, leading to an abnormal amount of polymerization. Steric hindrance of the fab segments normally limits the amount of polymerization of IgA. Bonner, et al proposes that a disturbance in the hinge region or an absence of fab. Similarly, decreased O-glycosylation might could destabilize the hinge region, allowing IgA to self associate. Likewise, destabilizing this region might make IgA susceptable to cleavage of fab fragments by bacterial proteases, leading to self aggregation and renal pathology.

:Because IgA has a high amount of glycosylation and a relatively large amount of flexibility, it has proven particularly difficult to crystallize in its intact form. Similarly, glycosylation and long linker regions between domains poses challenges to the crysatallization of the secretory component. Alternative techniques employed in these these studies included xray, neutron scattering analysis, analytical ultracentrifugation, and constrained modeling. Details provided in crystallographic studies – like disulfide bond, glycosylation residues and sites, detailed visualization of binding interaction – are absent in these results. Because of the limiting resolution of these models, many details concerning the binding residues and residue interactions are left unknown. Therefore, numerous questions are left unanswered, some of which are listed below. <ref name="eight"/>, <ref name="ten"/>, <ref name="five"/>, <ref name="seven" />

== Questions Unasnwered (a few of many)==

:Why does IgA2 lack as robust an effector function in binding to Fcalpha?

:: Crystal Structure of a Ligand-Binding Domain of the Human Polymeric Ig Receptor, pIgR [[1XED]]