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Digestion of IgA

Site-specific Digestion of Human IgA

IgASAP is a family of IgA-digesting enzymes. IgASAP Sub1 is an IgA1-specific enzyme that digests human IgA1 at one specific site above the hinge, and IgASAP Sub1+2 specifically digests IgA1 and IgA2m1 above the hinge. Both enzymes generate intact and homogenous Fab and Fc fragments, which allows for middle-level analysis of IgA.


 
IgA is the second most abundant antibody type in the human body and more IgA is produced daily than all other isotypes combined, making IgA an essential component of the immune system. Secretory IgA, found in mucous secretion, usually presents in dimeric form connected by a joining chain, the J-chain, and carries a secretory component. In association with mucous membranes, IgA acts by blocking foreign material from interacting with the epithelium in these highly exposed areas. IgA is also the second most prevalent immunoglobulin in serum, where it is most commonly present in monomeric form and triggers immune response by binding the Fc-receptor FcαRI.

There are two subclasses of human IgA, IgA1 and IgA2, and three allelic forms of human IgA2 (A2m1, A2m2 and A2m3). The IgA2 allotypes show variations in distribution among different ethnic groups and populations worldwide. The prevalence of the A2m1 allotype is highest in Caucasian whereas the A2m2 allotype is more common in populations of African and Asian descent. IgA1 is the dominant subclass in serum while the distribution in mucosal secretion is more even. The key role of IgA in the immune system makes it an interesting alternative to explore in therapeutic settings, creating the need for efficient tools for characterization and analysis.

IgASAP Digestion

Site-specific Digestion of Human IgA1

IgASAP Sub1 digests human IgA1 at one specific site above the hinge, generating Fab and Fc fragments, which allows for middle-level analysis of IgA1. To demonstrate the site-specificity of IgASAP Sub1, a commercially available human myeloma IgA1 was digested with IgASAP Sub1 Lyophilized for 1 hour at 37°C and compared to undigested IgA1.

As IgA1 carries multiple N- and O-glycans, the samples were treated with OmniGLYZOR Kit, including the additional deglycosylation step under denaturing conditions using PNGase F Lyophilized to remove all N-glycans, to facilitate the data interpretation. The samples were analyzed with LC-MS (Fig. 1).

The digestion using IgASAP Sub1 is fast, easy, and carried out under physiological conditions, and the specificity of the enzyme prevents overdigestion of IgA1. Deconvoluted mass spectra of N- and O-deglycosylated heavy chain (HC) of the IgA1 from human myeloma reveals a highly heterogenous material. After digestion using IgASAP Sub1, the heterogeneity of the HC material follows to the digested Fc part. It derives from several C-terminal truncations and a remaining extended O-glycan, not removed by OmniGLYZOR. The Fd fragment is homogenous and the acquired masses confirms the single digestion site at VPSTPP / TPSPST (aa 108/109 of constant heavy chain acc. to Uniprot p01876).

Despite the heterogenous starting material, the data clearly shows the single digestion site in the upper hinge region of human IgA1 and emphasizes the advantages of using a middle-level workflow to generate high resolution mass data for such complex samples.

Figure 1. Site-specific digestion of IgA1. a) Schematic illustration of the workflow. Deconvoluted mass spectra of N- and O-deglycosylated b) heavy chain (HC) of a commercially available IgA1 from human myeloma and the corresponding c) scFc fragment and d) Fd fragment after digestion using IgASAP Sub1 for 1 hour at 37°C. The samples were separated by reversed-phase chromatography (ACQUITY Premier Protein BEH C4, 300 Å, 1.7 µm 2.1 x 100 mm, Waters™) and analyzed with ESI-QTOF MS (Bruker Impact II).

IgASAP Digestion

Site-specific Digestion of Human IgA2m1

IgASAP Sub1+2 digests human IgA1 and IgA2 of allotype A2m1 at one specific site above the hinge. The above-hinge digestion results in the generation of Fab and Fc fragments, which allows for middle-level analysis. The reaction is carried out under physiological conditions, and the specificity of IgASAP Sub1+2 prevents overdigestion of the substrates.

To demonstrate the site-specificity of IgASAP Sub1+2, a commercially available recombinant human IgA2m1 was digested with IgASAP Sub1+2 Lyophilized overnight at 37°C, and compared to the undigested antibody. To facilitate data interpretation, the samples were treated with PNGaseF Lyophilized under denaturing conditions for removal of all Fab and Fc N-glycans. The denatured samples were analyzed with LC-MS (Fig. 2), and the acquired masses of the homogenous scFc and Fd fragments generated from IgASAP Sub1+2 hydrolysis confirm the single digestion site on human IgA2m1 at VTVPCP/VPPPPP (aa 102/103 of constant heavy chain acc. to Uniprot A0A0G2JMB2). The high-resolution mass data demonstrates the value of using middle-level analysis for characterization of the IgA2m1 antibody.

Figure 2. Site-specific digestion of IgA2m1. a) Schematic illustration of the workflow. Deconvoluted mass spectra of N-deglycosylated b) heavy chain (HC) of a commercially available recombinant IgA2m1 and the corresponding c) scFc fragment and d) Fd fragment after digestion using IgASAP Sub1+2 in an overnight reaction at 37°C. The samples were separated by reversed-phase chromatography (ACQUITY Premier Protein BEH C4, 300 Å, 1.7 µm 2.1 x 100 mm, Waters™) and analyzed with ESI-QTOF MS (Bruker Impact II).

IgASAP Digestion

Isotype and Subclass Specificity of the IgASAP Enzymes

To establish the specificity of the IgASAP enzymes, all four subclasses of human IgG along with IgM and IgA from different sources were incubated with IgASAP Sub1 Lyophilized for 1 hour at 37°C, and IgASAP Sub1+2 Lyophilized overnight at 37°C, before analysis with reducing SDS-PAGE (Fig. 3). All IgA substrates were specifically hydrolyzed (Fig. 3a) while no digestion was observed for any of the IgG subclasses or IgM (Fig.3b).

IgA1 constitutes 80-90% of IgA in human serum and approximately 40% of IgA in mucosal secretions (e.g. colostrum), while IgA2 makes up for the remaining percentages. This corresponds well with the digestion levels of IgA observed in Figure 3a. Some undigested IgA2 of the pooled serum IgA is expected from the IgASAP Sub1+2 digestion-resistant allelic forms A2m2 and A2m3. The IgA purified from colostrum is clearly from donors carrying A2m1 genetic variant as all IgA2 is completely hydrolyzed by IgASAP Sub1+2. The slightly larger IgA1 Fc fragments from IgASAP Sub1+2 digestion as compared to IgASAP Sub1 digestion results from the six amino acids difference in digestion site.

This demonstrates the high specificity of IgASAP Sub1 towards the IgA1 subclass, the high specificity of IgASAP Sub1+2 towards both IgA1 and IgA2m1 subclasses, and the ability of both enzymes to digest secretory and serum type IgA.

Figure 3. Isotype and subclass specificity of IgASAP Sub1 and IgASAP Sub1+2. The activity of the enzymes was assessed using human antibodies of various isotypes and subclasses. a) IgA substrates were digested as follows: IgA1 components were digested by both IgASAP Sub1 and IgASAP Sub1+2, IgA2m1 components were digested by IgASAP Sub1+2 only, and other IgA2 components (IgA2m2, IgA2m3) were not digested by either enzyme. b) The IgASAP enzymes did not show activity on either IgG or IgM components. All samples were treated at 1U/µg and incubated with IgASAP Sub1 Lyophilized for 1 hour at 37°C, or with IgASAP Sub1+2 Lyophilized overnight at 37°C, before analysis on reducing SDS-PAGE.

IgASAP Digestion

Subclass-specific Digestion of IgA in Serum

IgA constitutes around 15% of the total amount of immunoglobulins in normal human serum. The ability to selectively digest IgA1 and IgA2m1 can be of great value in research and diagnostics as well as in bioprocessing and therapeutic applications. The specificity of the IgASAP enzymes make them excellent tools for such isotype-specific digestion of IgA in human serum samples or other complex sample matrices.

Here, human serum was incubated with IgASAP Sub1 Lyophilized for 1 hour at 37°C and IgASAP Sub1+2 Lyophilized overnight at 37°C, before analysis with reducing SDS-PAGE and western blot (Fig. 4). IgASAP Sub1 specifically digests IgA1 in serum while leaving IgA2 and the other components of the serum intact. IgASAP Sub1+2 elicits a higher degree of digestion in the serum samples as the IgA2m1 fraction is also hydrolyzed. Remaining intact IgA2 in the pooled human serum after IgASAP Sub1+2 treatment is from the digestion-resistant allelic forms IgA2m2 and IgA2m3.

This shows the high subclass specificity of the IgASAP enzymes and demonstrates the possibility to use the products in complex biological samples such as serum and plasma without the risk of off-target digestion.

Figure 4. IgA-specific activity of IgASAP Sub1 and IgASAP Sub1+2 in human serum. Human serum was incubated with IgASAP Sub1 for 1 hour at 37°C or IgASAP Sub1+2 overnight at 37°C. The digested samples and control samples of human IgA1 (myeloma) and IgA2m1 (recombinant) subclasses were separated on three reducing SDS-PAGE gels. One of the gels was stained with Coomassie blue (left), and the other two gels were transferred to membranes for Western blotting. After blocking of the membranes in a casein solution, alkaline phosphatase conjugated α-human IgA1 Fc antibody (middle) or alkaline phosphatase conjugated α-human IgA2 Fc antibody (right), respectively, was added to the membranes and incubated for 1 hour at room temperature. The membranes were thoroughly washed before addition of the chromogenic substrate BCIP/NBT. IgA1 in serum was digested by both IgASAP Sub1 and IgASAP Sub1+2, while IgA2m1 was specifically digested by IgASAP Sub1+2. The remaining IgA2 in the pooled serum following IgASAP Sub1+2 digestion is from the digestion-resistant IgA2m2 and IgA2m3 forms. The low anti-IgA1 response of the serum control sample in the middle membrane is likely due to interference of other proteins.

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