Created by Elise Huppert

      Monoclonal Antibody IgG2a (PDB ID: 1IGT), also known as Mab231, is an immunoglobulin native to Mus musculus weighing 148.7 kDa with pI of 7.31, indicating near neutrality at biological pH (1,2). It was isolated in its entirety and its structure evaluated through crystallization at 18° C in polyethylene glycol 3350 (4%) between a pH of 7.8 and 8.2. This process yielded triclinic crystals, meaning they were relatively unorganized around any particular axis and subject to no strict symmetry (3). Immunoglobulins, also known as antibodies, are proteins responsible for recognition of foreign substances based on chemical labels called antigens (4). The term immunoglobulin represents a large family of closely related proteins featuring variations reflective of species and target antigen. Mab231, though endemic to mice, has been shown to enhance chemotherapy treatment in cases of canine lymphoma and non-Hodgkins lymphoma (3). The antibody has been shown to bind to tumor cells specifically and aggressively, without affecting normal cells, and has therefore become a standard and effective supplement to chemotherapy in treatment of these illnesses (5). It inhibits the development of tumors by triggering apoptosis in lymphoma cells and potentially by promoting creation of lymphoma specific T cells primed to recognize Mab antigens in what has been aptly called, “the vaccinal effect,” (5). Thorough analysis of IgG2a’s structure and function may increase understanding of lymphoma as well as the mechanisms available for developing treatment. 

      The broad class of antibody proteins share a general structure. They are typically tetramers consisting of two identical heavy (H) and two identical light chains (L) which compose two Fabs and one Fc functional fragments in a distinct Y-shape. Each heavy chain contains four domains: the variable domain (VH), and three constant domains (CH1, CH2, CH3). Similarly, the light chain has a variable domain (VL) and one constant domain (CL) (4). These variable domains are defined as the first 108 residues from the chain’s N terminus, and determine a single antibody’s specific function, as well as endowing the entire family with its power to recognize increasingly diverse substrates. This results in a vast family of closely-related, yet functionally different proteins (6). The 12 domains pair VH:VL, CL:CH1, CH2:CH2, and CH3:CH3 to form the Fab and Fc fragments, as well as a hinge region connecting them (4). 

      Mab231 secondary structure consists primarily of antiparallel β-sheets organized into twelve β-barrels forming the Fabs and Fc and random coils connecting them at the hinge. It contains few α-helical segments, which is typical of immunoglobulins (3). The distinct chains and resulting domains are held in place by multiple intra- and inter-molecular disulfide bonds, most importantly in the hinge, which links the 3 functional units and enables flexibility in the Fabs (6). These disulfide bonds occur between Cys-237, Cys-240, and Cys-242 of either chain. These disulfide bonds, in junction with the β-barrel secondary structure and the separation of the Fab and Fc functional fragments, create the characteristic immunoglobulin Y shape (3,6).

       The Fc region houses the interface between the CH2 and CH3 domains, which is essential to antigen binding. This Fc binding allegedly accounts for Mab231’s specificity to cancerous lymphocytes, mitigating toxicity to healthy cells in treatment of canine and non-Hodgkins lymphoma (5). A known substrate for this and similar immunoglobulins, Protein A, contains 5 domains which form 3 α-helices and interacts with this interface (4, 7).  Attraction between two of these helices and IgG2a’s third antiparallel β-sheet dock Protein A (7). In addition, residues Thr-264- Thr-267, Ile-330, and Glu-465 participate in specific intermolecular interactions that enhance binding (3). Thr-264 – Thr-267 define the first loop of CH2, all four residues of which participate in protein-substrate H bonds. Considering its substantial contribution to function, it is unsurprising that the 264-267 loop is consistently conserved and positioned in space across multiple observations. Within the CH2 domain, loops spanning from residues 278-286, 313-318, and 244-350 contribute to antigen docking. The CH2/CH3 interface is also responsible for IgG proteins’ active role in the classical complement pathway for threat recognition and neutralization. In particular Glu-337, Lys-339, and Lys-341 bind directly to the antigen via ionic attraction to specific C1q antigen sites, which activates the complement pathway ultimately induces lysis in the foreign cell (8). These three sites were conserved in IgG2a, and effectively triggered the complement pathway with C1q (3). 

       It is likely that Protein A also binds at the Fabs, but the particular mechanism by which this occurs remains unclear. Another known substrate, Protein G, binds to the Fab CH1 region first via complementary antiparallel β-sheet interaction and complementary attractions between side chains and the backbone at the Fabs (4). Residues Ala-118, Thr-121, and Ser-215 – Val-219 in particular contribute to tight binding due to H bonds between the side chains of very polar residues and the backbone of Protein G. This ligate may also form a similar α-helix complex at the Fc as Protein A rotated 180°, but this relationship is less defined (7). Both the Fc and Fabs are crucial to maximizing recognition, though the importance of each varies with antigen. This suggests that the binding of Fc fragment to its respective substrates promotes subsequent binding activity within the Fabs because it allows them the flexibility they need to bind with the stability they need to be effective. Under this assumption, the hinge region could be likened to a stem with two swivels rather than a true hinge (3).

       Antibody IgG2a exists in nature as an N-linked glycoprotein, and suffers diminished function in the absence of its ligand. The carbohydrate ligand is in fact two identical but independent oligosaccharides consisting of an N-acetyl-D-glucosamine, α- and β-D-mannose core whose branches include α- and β-L-fucose, and β-D-galactose (1, 3). The amino sugar is covalently linked to Asn-314 by an amide bond, though sugar protein contact occurs at a number of locations. α- and β-L-fucose act as H bond acceptors at their C-5 hydroxyl moieties to Tyr-313 on either heavy chain (1, 3). β-D-galactose participates in three distinct H bonds: it accepts a H from the Lys-259 amino R chain at its pyranose O, accepts the Thr-273 hydroxy H at its C-6 hydroxyl, and donates its C-5 hydroxy H to Asp-262 (1). There is another known saccharide-protein contact at Phe-256, which, unlike the previous residues, cannot participate in polar interactions and instead provides a docking surface on its flat aromatic ring (3). As mentioned, the oligosaccharide ligand is essential to function, despite the fact that it does not directly contact the antigen binding site. It likely induces a conformational change to promote effective binding (3). 

       Mab231 is a good example of typical immunoglobulin structure and function, but it also shares many characteristics with antibodies that diverge from the common form. For example, NGF monoclonal antibody AD11 (PDB ID: 1ZAN), an immunoglobulin found in Rattus norvegicus, appears unlike the classic immunoglobulin described here. Upon rudimentary examination, Mab231 appears much larger and features three obvious subunits in comparison to AD11’s two, and AD11 does not conform to the classic Y shape (1). However, the two proteins share nearly identical sequences, domains, and functions. Their parallels can be confirmed through use of the Dali Server and NCBI Psi-Blast, two online resources with distinct methods for the quantification of protein similarity. The Dali Server quantifies protein homology in tertiary structure by comparing intramolecular distances and delivers a Z-score which increases directly with fold overlap. A statistically significant Z-score is greater than 2, and the Z-score between IgG2a and AD11 is 27.7, indicating substantial overlap in tertiary structure (9). In contrast, Psi-Blast compares peptides by primary structure, or amino acid sequence, and delivers an E-score proportional to the, “gaps,” or lapses in shared sequence. Therefore, an E-score less than 0.05 indicates considerable similarity, and IgG2a and AD11 had an E-score equal to 4 x 10-120 (10).  This program again affirms the relationship between the two antibodies, despite the latter’s divergence from the typical immunoglobulin Y-shaped motif. 

      The AD11 immunoglobulin opposes the nerve growth factor (NGF), which is involved in modulating a pain response. AD11 and IgG2a differ primarily in that AD11 contains only one Fab region connected to the Fc, which causes the obvious shape divergence (11). Still, the Psi Blast and Dali Server results suggest that the remaining regions are very close in sequence and structure. Where the light and heavy chains of IgG2a are 214 and 444 respectively, the light and heavy chain of AD11 are 214 and 224 residues long, and it has only one of each (1). These chains fold into six loops, three light and three heavy, which compose the Fab sector, which is responsible for the majority of docking and neutralizing the NGF antigen. The heavy loops are made flexible by high Gly content, which allows the antibody to snare the antigen from a variety of orientations. This Gly content is offset by a polar region in the H1 loop from Asn-31 to Asn-35 to increase solubility. There also exists direct Fab/NGH H bonding between Tyr-30, Asn-50, and Tyr-191 in the light chain and Asn-30 and Tyr-98 in the heavy chain. This high concentration of H bonds within a relatively short peptide length contributes to tight binding and effective NGF neutralization. Like IgG2a, AD11 is constituted of primarily of β-sheets, but unlike IgG2a it is not a glycoprotein. Rather, AD11 is associated with a chloride ion H bonded to Asn-120 (11).

       Monoclonal Antibody IgG2a is an example of paradigm immunoglobulin structure and function. It is emblematic of even divergent immunoglobulins and its application to treatment of canine lymphoma promises improved possibilities for human medicine.