CD99L2 Antibody

What is CD99L2 and what is its role in leukocyte migration?

CD99L2 is a highly glycosylated 52 kDa type-1 transmembrane protein that shares approximately 32% amino acid identity with CD99. It plays a crucial role in leukocyte transendothelial migration (TEM), particularly at a specific step between those regulated by PECAM and CD99. CD99L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. The protein regulates a unique sequential step in TEM, rather than operating in parallel or redundantly with other molecules such as PECAM and CD99. Functionally, CD99L2 promotes transmigration by recruiting the lateral border recycling compartment (LBRC) to sites of TEM, specifically downstream of PECAM initiation .

In experimental models, inhibiting CD99L2 using function-blocking antibodies significantly reduces leukocyte recruitment to inflammation sites. Similarly, CD99L2 knockout mice demonstrate an inherent defect in leukocyte transmigration during inflammatory responses. These findings highlight the protein's critical importance in the multi-step process of leukocyte extravasation during inflammation .

What are the key characteristics of commercially available CD99L2 antibodies?

Commercial CD99L2 antibodies are available in various formats, including polyclonal and monoclonal variants. For example, antibody 13732-1-AP is a rabbit polyclonal IgG that targets human CD99L2 in Western Blot and ELISA applications. This particular antibody has an observed molecular weight detection of approximately 40 kDa, despite the calculated molecular weight of 28 kDa (262 amino acids), likely due to post-translational modifications such as glycosylation .

The recommended dilution ranges for these antibodies vary by application: typically 1:500-1:2000 for Western Blot analysis. Most CD99L2 antibodies are provided in liquid form, purified through antigen affinity methods, and stored in PBS with preservatives such as sodium azide and glycerol. Proper storage conditions generally require -20°C, with stability guaranteed for approximately one year after shipment .

How does CD99L2 compare structurally and functionally to CD99?

CD99L2 shares approximately 32% amino acid identity with CD99, suggesting some structural similarities but distinct functions. Both proteins are type-1 transmembrane proteins expressed on leukocytes and endothelial cells. Functionally, both CD99 and CD99L2 are involved in the process of leukocyte transendothelial migration, but they regulate different sequential steps in this process .

What are the optimal protocols for CD99L2 antibody validation in human samples?

For effective CD99L2 antibody validation in human samples, researchers should employ a multi-method approach. Begin with Western blot analysis using HUVEC (Human Umbilical Vein Endothelial Cells) as positive controls, as these cells demonstrate reliable CD99L2 expression . The recommended antibody dilution range is 1:500-1:2000, but optimization for specific experimental conditions is advisable.

For validation experiments, include appropriate positive and negative controls. HUVEC cells serve as excellent positive controls, while cells known to lack CD99L2 expression or CD99L2 knockdown cells can function as negative controls. For knockdown validation, researchers can use shRNA approaches targeting the 3' untranslated region of CD99L2 (e.g., 5'-CCGGCGATGTCAAGAACGAGCATCAAATCGATTTGATGCTCGTTCTTGACATCGTTTTT-3'), which have been successfully implemented in previous studies .

Additionally, immunofluorescence staining can confirm the expected localization pattern at endothelial cell borders. For optimal results, fixation methods should be carefully selected as they can affect antibody binding and epitope accessibility. Paraformaldehyde fixation (typically 4%) followed by permeabilization with a mild detergent is often suitable for membrane proteins like CD99L2 .

What are the recommended approaches for CD99L2 knockdown studies?

For CD99L2 knockdown studies, short hairpin RNA (shRNA) approaches have proven effective. Based on published methodologies, researchers should design shRNA constructs targeting specific regions of CD99L2 mRNA. For instance, successful knockdown has been achieved using shRNA targeting the 3' untranslated region with the sequence: 5'-CCGGCGATGTCAAGAACGAGCATCAAATCGATTTGATGCTCGTTCTTGACATCGTTTTT-3' .

The shRNA construct should be cloned into an appropriate vector system, such as pENTR4 containing a hU6 promoter. For efficient delivery to endothelial cells, adenoviral vector systems have been successfully employed. The cloning procedure involves transferring the shRNA construct from the entry vector (e.g., pENTR4) to an adenoviral destination vector (e.g., pAd/PL-DEST) via recombinase reaction using LR Clonase .

For virus production, transfect the recombined plasmid into 293A cells using Lipofectamine 2000 according to standard protocols. After cell lysis, collect cells and media, and subject them to freeze/thaw cycles (typically three) to release viral particles. Remove cell debris by centrifugation (3000 rpm, 30 minutes, 4°C), filter the supernatant through a micron filter, and store aliquots at -80°C. Validate knockdown efficiency through Western blot and functional assays before proceeding with experimental analyses .

How should researchers design transmigration assays to study CD99L2 function?

When designing transmigration assays to study CD99L2 function, researchers should consider both in vitro and in vivo approaches. For in vitro studies, transwell systems using primary human endothelial cells (such as HUVECs) grown to confluence on permeable supports are recommended. Researchers should verify the integrity of the endothelial monolayer before experiments, typically using electrical resistance measurements or permeability assays with fluorescent tracers .

To analyze CD99L2's specific role in the TEM cascade, sequential blocking experiments are valuable. This involves using specific blocking antibodies against multiple TEM-related molecules (PECAM, CD99, and CD99L2) individually and in combination. Such experiments can reveal whether CD99L2 functions at a distinct step compared to other molecules. Time-lapse microscopy during these assays can provide valuable insights into the specific stage at which leukocyte migration is arrested when CD99L2 is inhibited .

For quantifying transmigration, fluorescent labeling of leukocytes prior to the assay allows for straightforward enumeration of cells that successfully migrate through the endothelial layer. Additionally, analyzing the distribution of arrested leukocytes (whether they are on the apical surface, engaged with junctions, or partially transmigrated) provides critical information about the specific stage of migration affected by CD99L2 inhibition or knockdown .

How does CD99L2 interact with the lateral border recycling compartment (LBRC) during transmigration?

The mechanism involves CD99L2-mediated signaling that helps direct the targeted recycling of LBRC to the sites where leukocytes are actively migrating through endothelial junctions. This recruitment provides additional membrane surface area and adhesion molecules needed to facilitate leukocyte passage. When CD99L2 function is inhibited, either through antibody blockade or genetic knockdown, this recruitment process is disrupted, resulting in arrested leukocyte transmigration .

To investigate this interaction experimentally, researchers can employ live-cell imaging with fluorescently tagged LBRC markers (such as PECAM-GFP) in combination with CD99L2 inhibition. Additionally, super-resolution microscopy techniques can provide detailed visualization of the spatial relationships between CD99L2 and LBRC components during active transmigration events. Quantitative analysis of LBRC recruitment in the presence and absence of functional CD99L2 can further elucidate the specific contributions of this protein to LBRC dynamics .

What are the differences in CD99L2 function between mouse and human systems?

In human systems, CD99L2 is constitutively expressed on leukocytes and at endothelial cell borders, similar to mice. Human CD99L2 functions in a homophilic interaction manner, with the protein on both leukocytes and endothelial cells contributing to successful transmigration. Mechanistically, human CD99L2 regulates a specific sequential step of TEM between PECAM and CD99, rather than operating in parallel or redundantly with these molecules .

These differences highlight the importance of careful experimental design when translating findings between species. Researchers should validate observations made in mouse models using human cells and tissues whenever possible, particularly when developing therapeutic strategies targeting CD99L2 .

How does CD99L2 deficiency affect neuroinflammation models?

CD99L2 deficiency has significant effects on neuroinflammation models, particularly in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Conditional Tie-2-Cre driven gene inactivation of CD99L2 interferes with EAE development by inhibiting diapedesis of leukocytes through the endothelial basement membrane of the blood-brain barrier (BBB) .

This inhibition of leukocyte entry into the central nervous system (CNS) results in amelioration of neuroinflammation. The mechanism appears to involve a specific blockade at the stage of transmigration where leukocytes need to overcome the endothelial basement membrane, suggesting that CD99L2 may play a particularly important role in this critical step of extravasation into the CNS .

For researchers studying neuroinflammation, these findings highlight CD99L2 as a potential therapeutic target. Experimental approaches to investigate this further might include the use of function-blocking antibodies against CD99L2 in EAE models, tissue-specific conditional knockout strategies, and detailed analysis of the leukocyte subsets most affected by CD99L2 deficiency in the context of CNS inflammation .

How can researchers address inconsistent results with CD99L2 antibodies in Western blot applications?

Inconsistent results with CD99L2 antibodies in Western blot applications can stem from several factors. First, sample preparation is critical - CD99L2 is a highly glycosylated protein with an observed molecular weight of approximately 40 kDa despite a calculated weight of 28 kDa. Deglycosylation treatments prior to SDS-PAGE can help resolve inconsistencies in band patterns. For optimal results, researchers should ensure complete denaturation of samples using appropriate buffers containing SDS and reducing agents, with heating at 95°C for 5 minutes .

The membrane transfer step is equally important. CD99L2, as a membrane protein, may require optimized transfer conditions. Using PVDF membranes rather than nitrocellulose and adjusting transfer parameters (longer transfer times or lower voltage) can improve detection. Additionally, blocking solutions containing 5% non-fat dry milk or BSA in TBS-T are typically effective, but optimization may be necessary .

For primary antibody incubation, researchers should titrate the concentration (recommended range 1:500-1:2000) and incubate overnight at 4°C for optimal results. If background issues persist, increasing the number and duration of wash steps or adding low concentrations of detergents to wash buffers can help. Finally, validation with positive controls (such as HUVEC cells) and negative controls (such as CD99L2 knockdown cells) is essential for confirming antibody specificity and troubleshooting inconsistent results .

What controls are essential for interpreting CD99L2 functional assays correctly?

For rigorous interpretation of CD99L2 functional assays, multiple controls are essential. Positive controls should include conditions known to demonstrate normal CD99L2 function, such as wild-type cells or tissues in transmigration assays. Negative controls should include CD99L2 knockdown or knockout models, as well as isotype control antibodies when using blocking antibody approaches .

Time-course studies are also critical for correct interpretation. Since CD99L2 functions at a specific step in the transmigration process, assessing the location of arrested leukocytes at various time points can provide valuable insights. This approach allows researchers to distinguish between defects in initial adhesion, early transmigration steps, or later diapedesis through the basement membrane .

Finally, complementation experiments, where CD99L2 expression is restored in knockout or knockdown models, serve as essential controls to confirm that observed phenotypes are specifically due to CD99L2 deficiency rather than off-target effects .

How should researchers account for strain-specific differences when studying CD99L2 in mouse models?

When studying CD99L2 in mouse models, accounting for strain-specific differences is crucial for accurate data interpretation. Research has demonstrated that the specific step at which CD99L2 functions in the transmigration cascade differs between mouse strains. In FVB/n mice, CD99L2 deficiency arrests leukocytes at a stage beyond the PECAM-dependent step, while in C57BL/6 mice, CD99L2 controls a different stage of leukocyte transmigration .

To account for these differences, researchers should first clearly document and report the specific strain used in all experiments. When comparing results across studies, strain differences must be explicitly considered as potential sources of discrepancy. Whenever possible, key experiments should be replicated in multiple strains to assess the generalizability of findings .

For mechanistic studies, researchers can leverage these strain differences as a tool to dissect the specific roles of CD99L2. By comparing the stage at which leukocyte transmigration is arrested in different genetic backgrounds, more nuanced understanding of CD99L2 function can be gained. Additionally, generating strain-specific knockout or knockin models may reveal genetic modifiers that interact with CD99L2 signaling pathways .

Finally, when translating findings from mouse models to human applications, researchers should acknowledge these strain-specific variations and conduct parallel experiments using human cells to validate key observations .

What is known about CD99L2's potential role in neurological disorders beyond EAE?

CD99L2's role in neurological disorders extends beyond experimental autoimmune encephalomyelitis (EAE), though this area remains underexplored. The protein's demonstrated ability to regulate leukocyte entry into the central nervous system by facilitating migration through the blood-brain barrier suggests potential relevance to various neuroinflammatory conditions. Research indicates that CD99L2 may function as a homophilic adhesion molecule and plays a role in helping leukocytes overcome the endothelial basement membrane, a crucial step in CNS infiltration during inflammatory processes .

The expression of CD99L2 on neuronal cells, in addition to leukocytes and endothelial cells, suggests possible direct roles in neuronal function or neuron-immune interactions that remain to be fully characterized. This neuronal expression pattern opens avenues for investigating CD99L2's potential involvement in neurodegenerative disorders where neuroinflammation is a component, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis .

How might CD99L2-targeting approaches be developed for therapeutic applications?

Developing CD99L2-targeting therapeutic approaches requires multiple strategies. Function-blocking antibodies represent a promising direction, as they have demonstrated efficacy in reducing leukocyte recruitment to inflammation sites in experimental models. These antibodies could be humanized and optimized for clinical applications, with careful attention to specificity and potential off-target effects. Additionally, small molecule inhibitors that disrupt CD99L2 homophilic interactions or interfere with its signaling pathways could provide alternative therapeutic options .

Another approach involves targeted delivery systems that can specifically inhibit CD99L2 at sites of inflammation while minimizing systemic effects. This might include nanoparticle-based delivery of CD99L2 inhibitors or siRNA/shRNA constructs to downregulate CD99L2 expression. For neuroinflammatory conditions, delivery systems capable of crossing or targeting the blood-brain barrier would be particularly valuable .

The development of soluble CD99L2-Fc fusion proteins, which could competitively inhibit CD99L2 homophilic interactions, represents another potential therapeutic strategy. Previous research has successfully generated such constructs by amplifying the extracellular domain of CD99L2 and inserting it into Fc-containing vectors. These fusion proteins could serve as decoys to prevent the normal function of membrane-bound CD99L2 during inflammatory processes .

Given CD99L2's sequential role in the transmigration cascade, combination therapies targeting multiple steps in this process might provide synergistic benefits while potentially allowing lower doses of individual agents to minimize side effects .

What techniques are emerging for studying CD99L2 interactions with other membrane proteins?

Emerging techniques for studying CD99L2 interactions with other membrane proteins include advanced imaging methods and molecular interaction assays. Super-resolution microscopy techniques such as stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy enable visualization of protein distributions and potential co-localization at nanometer resolution, far exceeding the capabilities of conventional confocal microscopy. These approaches can reveal precise spatial relationships between CD99L2 and other transmigration-related proteins during active leukocyte migration .

Proximity ligation assays (PLA) offer another powerful technique for detecting potential protein-protein interactions in situ. This method can demonstrate close associations (typically <40 nm) between CD99L2 and other proteins in fixed cells or tissues, providing evidence for potential interactions. For more direct assessment of physical interactions, techniques such as Förster resonance energy transfer (FRET) between fluorescently labeled proteins can demonstrate molecular proximity at even smaller scales (typically <10 nm) .

Biochemical approaches including co-immunoprecipitation optimized for membrane proteins (using appropriate detergents and crosslinking strategies) can identify stable interactions. For more transient or weak interactions, chemical crosslinking followed by mass spectrometry (XL-MS) can capture and identify interaction partners. Additionally, protein complementation assays, such as split-GFP or split-luciferase systems, allow for monitoring of protein interactions in living cells .

For high-throughput screening of potential interaction partners, techniques such as BioID or APEX2 proximity labeling, where proteins in close proximity to CD99L2 become biotinylated and can subsequently be isolated and identified by mass spectrometry, are increasingly valuable for mapping the CD99L2 interactome .