V region carbohydrate and antibody expression.

Area of Science:

• Immunology
• Glycobiology
• Structural Biology

Background:

• N-linked glycosylation in immunoglobulin (Ig) variable (V) regions is common and influences antigen binding.
• Carbohydrates in the V region of Ig heavy (H) chains can modulate antigen binding affinity.
• The V region of Ig H chains, particularly complementarity-determining regions (CDRs), plays a critical role in antibody specificity.

Purpose of the Study:

• To investigate the glycosylation status of a high-mannose carbohydrate within the CDR2 of a murine anti-dextran V(H).
• To determine the impact of V region glycosylation on antibody folding, trafficking, and secretion.
• To explore how V region carbohydrate modifications affect antibody function and production.

Main Methods:

• Analysis of N-linked carbohydrate processing in a murine anti-dextran V(H) containing a CDR2 glycan.
• Production of murine-human chimeric antibodies in various cell types to assess glycan processing.
• Engineering of chimeric antibodies by replacing CDR2 regions to study the effects of glycosylation site insertion.

Main Results:

• The V(H) CDR2 glycan consistently remained high mannose, irrespective of cell type or the presence of other glycosylation sites.
• Introducing the V(H) CDR2 glycosylation site into an anti-dansyl V(H) led to H chain retention in the endoplasmic reticulum.
• Antibodies with inappropriate V region glycosylation failed to traffic to the Golgi apparatus, indicating impaired secretion.

Conclusions:

• Inappropriate glycosylation within antibody V regions can lead to misfolding and impaired secretion.
• The location and type of N-linked carbohydrate addition in CDRs can significantly impact antibody production efficiency.
• Aberrant glycosylation sequences arising from gene rearrangement or somatic hypermutation may result in non-functional antibodies.