CD23 Human, Sf9

What is human CD23 and what is its role in immune regulation?

Human CD23 (also known as FcεRII) is a 45-kDa type II transmembrane glycoprotein that functions as the low-affinity receptor for IgE. This protein is primarily expressed on B lymphocytes and monocytes, but also appears on T lymphocytes, eosinophils, platelets, Langerhans cells, follicular dendritic cells, and neutrophils . CD23 is spatially associated with the major histocompatibility complex (MHC) Class II antigen and contributes to allergic inflammation through allergen presentation to T cells and regulation of IgE responses .

On B lymphocytes specifically, CD23 expression increases upon activation and is subsequently lost during differentiation into antibody-secreting plasma cells. This dynamic regulation suggests its important role in B-cell maturation processes . CD23 can also be released from cell surfaces as soluble fragments (sCD23), which have been shown to possess cytokine-like activities .

What are SF9 cells and why are they preferred for CD23 expression?

SF9 cells are an insect cell line derived from Spodoptera frugiperda (fall armyworm) that are widely used for recombinant protein expression, particularly with baculovirus expression systems. Researchers prefer these cells for CD23 expression for several reasons:

• SF9 cells can produce properly folded and monomeric forms of CD23 derivatives

• They support the expression of different CD23 constructs spanning various functional regions

• The baculovirus-SF9 system yields relatively high amounts of biologically active protein

• Full-length 45-kD CD23 expressed in baculovirus-infected SF9 cells maintains its ability to bind to CD23 receptors on B-cell lines and monocytic cells

The recombinant proteins produced in this system have been successfully used as immunogens for antibody development and for functional studies investigating CD23's biological activities .

What are the structural components of human CD23 and how are they represented in research constructs?

Human CD23 contains several distinct structural domains with specific functions:

• A short cytoplasmic N-terminal domain

• A transmembrane region

• An extracellular stalk region containing a single N-linked glycosylation site at asparagine 63 (N63)

• A C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs)

For research purposes, several CD23 constructs have been developed for expression in SF9 cells:

• CD23A: Represents the full extracellular domain (amino acids 48-321), including both stalk and head regions

• CD23B: Identical to CD23A but with an N63Q substitution that eliminates the N-linked glycosylation site

• CD23C: Contains only the head domain (amino acids 150-321)

• CD23D: A smaller portion of the head domain (amino acids 156-298)

The 25-kD soluble fragment of CD23 (corresponding to amino acids 150-321) has been expressed in both baculovirus and E. coli systems for functional studies .

How does the stalk region of CD23 influence IgE binding properties?

Research has demonstrated that the stalk region of CD23 plays a critical and direct role in IgE binding. This finding has significant implications for experimental design when studying CD23-IgE interactions:

The stalk region (approximately amino acids 48-150) forms a coiled-coil structure that facilitates oligomerization of CD23 molecules, which in turn affects the avidity of CD23 for IgE . Constructs containing both the stalk and head regions (like CD23A and CD23B) demonstrate different IgE binding properties compared to head-only constructs (CD23C and CD23D) .

The N-linked glycosylation at asparagine 63 in the stalk region also modulates IgE binding, as evidenced by the different properties of CD23A (with glycosylation) versus CD23B (with the N63Q mutation that eliminates glycosylation) . This suggests that both the structural integrity of the stalk and its post-translational modifications are important determinants of CD23's interaction with IgE.

Researchers studying CD23-IgE interactions should consider these factors when designing constructs and interpreting binding data.

How does soluble CD23 (sCD23) affect cell survival and apoptosis?

Soluble CD23 (sCD23), particularly the 25-kD species, functions as a multifunctional cytokine that can significantly impact cell survival:

• sCD23 sustains the growth of low cell density cultures of the human pre-B-acute lymphocytic leukemia cell line SMS-SB, with no other tested cytokines able to induce this effect

• Flow cytometric analysis reveals that sCD23 prevents apoptosis in SMS-SB cells in a concentration-dependent manner, with 100 ng/mL sCD23 reducing the percentage of late apoptotic cells from approximately 25% to 6%

• Mechanistically, sCD23 maintains bcl-2 expression in cells cultured at low density at levels equivalent to those observed in higher-density cultures

• Interestingly, SMS-SB cells do not express the known receptors for CD23 (CD21, CD11b-CD18, or CD11c-CD18), yet they specifically bind CD23-containing liposomes. This binding is inhibited by anti-CD23 but not by anti-CD21 or anti-CD11b/c antibodies, suggesting the presence of a novel CD23 receptor on these cells

These findings highlight sCD23's potential role in B-cell development and leukemia progression, offering potential therapeutic targets for intervention.

What are the species-specific differences in CD23 function?

CD23 exhibits remarkable species-specific differences in binding capabilities and function:

Human CD23 differs significantly from CD23 in other mammalian species in its glycan-binding capabilities. While CD23 in most mammalian species contains residues required to form a sugar-binding site, human CD23 shows no sugar binding activity in experimental settings .

Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis demonstrate that cow and mouse CD23 bind to mannose, GlcNAc, glucose, and fucose, as well as to glycoproteins bearing these sugars in nonreducing terminal positions . Crystal structures of cow CD23 CRD in the presence of various sugar ligands reveal an open binding site where most interactions occur with a single terminal sugar residue .

Mouse CD23 shows a similar pattern of monosaccharide and glycoprotein binding as cow CD23, but with weaker binding affinity . The absence of sugar-binding activity in human CD23 correlates with accumulated mutations in the CD23 gene (FCER2) in the primate lineage, resulting in the loss of key sugar-binding residues .

What methodological approaches can resolve contradictions in CD23 receptor interaction data?

Resolving contradictions in CD23 receptor interaction data requires sophisticated experimental approaches:

When studying novel CD23 interactions, researchers should employ multiple complementary techniques. For example, the interaction between CD23 and SMS-SB cells (which lack known CD23 receptors) was demonstrated through several approaches: CD23-containing liposomes specifically bound to SMS-SB cells, this binding was inhibited by anti-CD23 but not by other antibodies, and functional studies showed biological effects of CD23 on these cells .

For comprehensive characterization of CD23 binding interactions, researchers should consider:

• Solid-phase binding competition assays to identify competing ligands

• Glycoprotein blotting experiments for analyzing interactions with complex glycoproteins

• Glycan array analysis for comprehensive screening of potential carbohydrate ligands

• Crystal structure analysis to elucidate the molecular basis of binding specificity

When contradictory results are obtained, systematic validation using different experimental systems can help identify method-specific artifacts. For instance, comparative studies of CD23 from different species (human, cow, mouse) using identical experimental conditions revealed consistent species-specific differences in binding properties .

What are optimal expression conditions for different CD23 constructs in SF9 cells?

Successful expression of functional CD23 constructs in SF9 cells requires optimization of several parameters:

For full-length CD23 (45-kD), baculovirus-infected SF9 cells have been successfully used with the protein subsequently affinity purified . The resulting protein maintains biological activity, as demonstrated by its ability to bind to CD23 receptors on B-cell lines and monocytic cells and its activity in human B-cell growth assays .

For the 25-kD soluble CD23 fragment (residues 150-321), both baculovirus-SF9 systems and E. coli expression systems have proven effective . The choice between these systems depends on the specific research requirements:

• Baculovirus-SF9 expression may preserve certain post-translational modifications

• E. coli expression typically yields higher protein amounts but lacks eukaryotic post-translational modifications

For constructs containing the N-linked glycosylation site at N63, SF9 cells provide appropriate glycosylation, although the glycan structures differ somewhat from mammalian cells. If studying the role of glycosylation is important, researchers can compare wild-type constructs (like CD23A) with glycosylation-deficient mutants (like CD23B with the N63Q substitution) .

The purified recombinant proteins can serve multiple purposes, including as immunogens for antibody development. For example, human recombinant protein fragment corresponding to amino acids 48-321 of human FCER2 produced in SF9 cells has been successfully used as an immunogen for antibody production .

How can crystal structure analysis inform the design of CD23 mutants with altered binding properties?

Crystal structure analysis provides critical insights for rational design of CD23 variants with modified binding properties:

Crystal structures of cow CD23 CRD complexed with α-methyl mannoside and GlcNAcβ1-2Man have revealed an open binding site where most interactions occur with a single terminal sugar residue . This structural information has several implications for designing CD23 mutants:

• Identification of key binding residues: By comparing crystal structures of CD23 from different species, researchers can identify the specific residues responsible for different binding properties. For instance, the loss of sugar-binding activity in human CD23 correlates with mutations in key residues in the sugar-binding site .

• Rational modification of binding specificity: Understanding the structural basis of CD23's interaction with different ligands allows for targeted mutagenesis to modify binding properties. For example, introducing specific mutations in human CD23 based on cow or mouse CD23 structures could potentially restore sugar-binding activity.

• Engineering of the stalk region: Crystal structure information combined with functional studies has highlighted the importance of the stalk region in IgE binding . Modifications to this region could potentially alter the oligomerization properties of CD23 and consequently its binding avidity for IgE.

• Development of therapeutic variants: Structure-guided modifications could lead to CD23 variants with enhanced or reduced binding to specific ligands, which could have therapeutic applications in allergy and inflammatory conditions.

What are the methodological approaches for studying CD23's role in preventing apoptosis?

Investigating CD23's anti-apoptotic effects requires sophisticated methodological approaches:

Flow cytometric analysis with dual staining (propidium iodide and Hoechst 33342) allows quantitative assessment of viable, apoptotic, and necrotic cells . This technique revealed that sCD23 reduces the percentage of apoptotic cells in low-density cultures of SMS-SB cells from approximately 25% to 6%, values comparable to those observed in normal-density cultures .

Morphological assessment by light microscopy provides complementary evidence of apoptotic characteristics. Cells cultured at low density in the absence of sCD23 exhibit spread morphology, adherence to tissue culture flasks, membrane blebbing, and cytoplasmic vacuolation - all hallmarks of apoptosis. Addition of sCD23 generates cells with morphology indistinguishable from normal-density cultures .

Molecular analysis of apoptosis-related proteins, particularly bcl-2, provides insights into the mechanism of action. SMS-SB cells cultured at low density possess low levels of bcl-2 protein, while addition of sCD23 maintains bcl-2 expression at levels equivalent to those observed in higher-density cultures .

To elucidate receptor interactions, researchers can employ liposome binding assays. SMS-SB cells specifically bind CD23-containing liposomes but not control liposomes, and this binding is inhibited by anti-CD23 but not by antibodies against known CD23 receptors . This methodological approach identified a potential novel CD23 receptor on these cells.