IgA N-Glycosylation Site Reduction Service

Enhanced Protein Stability

Glycosylation sites can sometimes be implicated in protein aggregation or degradation. Reducing N-Glycosylation in specific regions, as observed in studies on human IgA2 where the absence of glycans in the Fc tail region led to higher-order aggregates, can be strategically managed to improve overall protein stability.

Modulated Protein Activity

The presence or absence of specific glycans can directly influence IgA's binding affinity, effector functions, and interactions with other biological molecules. Targeted reduction allows for the optimization of these functional attributes, for instance, in designing optimal recombinant IgA molecules for targeted mucosal protection.

Optimized Protein Folding and Secretion

Especially relevant in recombinant protein production systems, uncontrolled or inappropriate glycosylation can interfere with proper protein folding or efficient secretion. Our precise N-Glycosylation site reduction strategies can overcome these challenges, ensuring high yields of properly folded and functional IgA.

Fundamental Research Insights

Beyond direct therapeutic applications, N-Glycosylation site reduction serves as an invaluable tool for researchers to elucidate the specific roles of individual glycan moieties in IgA's complex biology, including its diverse N-glycan compositions and body fluid-specific variations.

Targeted Glycoengineering

The cornerstone of our approach involves the precise modification of the IgA protein at the genetic level.

Functional Validation

Ultimately, the impact of N-Glycosylation site reduction on IgA's biological function is paramount. We offer a range of functional assays to confirm that the engineered IgA retains or exhibits enhanced desired activities, such as antigen binding, interaction with Fc receptors, or mucosal transport.

Genetic Engineering

Beyond single-site mutations, we can engineer IgA constructs with fewer or strategically altered N-Glycosylation sites by modifying the protein's gene. This allows for the creation of novel IgA variants with customized glycosylation profiles.

Site-Directed Mutagenesis

The most common and effective method involves mutating the asparagine (Asn) residue within the N-Glycosylation consensus sequence (AsN-X-Ser/Thr) to another amino acid, typically glutamine (Gln). This mutation abrogates the glycosylation motif, resulting in the complete loss of glycan attachment at that specific site. Our extensive experience ensures the strategic selection of mutation sites to achieve desired structural and functional outcomes.

Recombinant IgA Expression and Purification

Following genetic modification, the engineered IgA variants are expressed in suitable host systems. Creative Biolabs leverages optimized expression platforms to ensure high-yield production of the modified IgA, followed by rigorous purification protocols to obtain highly pure protein for subsequent analysis and validation.

Advanced Glycan Analysis and Characterization

The success of N-Glycosylation site reduction hinges on accurate verification of the altered glycan profile and its impact on protein structure and function. Creative Biolabs employs an array of sophisticated analytical techniques:

• Nano-HPLC-chip/TOF-MS (Nano-High-Performance Liquid Chromatography-chip/Time-of-Flight Mass Spectrometry): This highly sensitive technique is routinely used for the comparative analysis of IgA N-glycan profiles, allowing us to precisely identify and quantify changes in glycan compositions resulting from site reduction. This mirrors the advanced methodologies used in studies characterizing diverse IgA N-glycan compositions.
• Small Angle X-Ray Scattering (SAXS): For structural insights, SAXS is invaluable. As demonstrated in studies on IgA2, SAXS can reveal the effect of glycans on protein structure, flexibility, and the formation of higher-order aggregates in solution. Combined with atomistic modeling, SAXS provides critical data on how N-Glycosylation site reduction impacts the overall conformation and dynamics of IgA.
• Biochemical and Biophysical Methods: We utilize a suite of techniques including Differential Scanning Fluorimetry (DSF), nanoDSF, and Circular Dichroism (CD) spectroscopy to assess the thermal stability, folding characteristics, and thermodynamics of the modified IgA. These methods provide crucial data on how N-Glycosylation site reduction influences the intrinsic stability and structural integrity of the protein.
• Size-Exclusion Chromatography (SEC): SEC is employed to evaluate the oligomeric state and purity of the engineered IgA, ensuring that the reduction in N-Glycosylation sites does not lead to undesirable aggregation or misfolding.

Therapeutic Antibody Development

Engineering IgA for improved stability, reduced immunogenicity, and enhanced effector functions in novel therapeutic antibodies. This includes the rational design of optimal recombinant IgA molecules for targeted mucosal protection against infectious agents or inflammatory conditions.

Vaccine Design and Adjuvants

Developing IgA-based vaccine candidates with optimized glycosylation to elicit more potent and durable mucosal immune responses.

Diagnostic Biomarker Discovery

Investigating the role of specific IgA glycoforms as biomarkers for disease diagnosis and prognosis. For example, our work can guide salivary SIgA-glycaN-based biomarker research for the diagnosis of oral cavity diseases (e.g., periodontitis, Sjögren's syndrome) and systemic conditions (e.g., pulmonary and kidney diseases, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, cancer, or infectious diseases).

Immunology Research

Gaining deeper insights into the fundamental roles of IgA glycosylation in immune regulation, pathogen recognition, bacterial attachment, pathogen decoy, and clearance mechanisms.

Autoimmune Disease Research

Studying the precise impact of aberrant IgA glycosylation in autoimmune conditions and developing strategies to normalize or counteract these pathological modifications.

Protein Engineering and Bioprocessing

Optimizing the production and stability of recombinant IgA and other glycoproteins in various expression systems.