CD81 Human

What is the molecular structure of human CD81?

Human CD81 (hCD81) possesses a canonical tetraspanin structure with four transmembrane domains and two extracellular loops (EC1 and EC2). Biophysical characterization using circular dichroism has revealed that CD81 has a highly-defined α-helical secondary structure, with approximately 77.1% α-helical content, which correlates remarkably well with the expected 75.2% based on protein sequence and crystal structure data of the second extracellular loop . The protein contains palmitoylation sites that can be modified post-translationally, affecting its localization and function in cellular membranes. Recent structural studies have begun to elucidate how CD81 organizes into tetraspanin-enriched microdomains, although differences exist between various tetraspanin family members .

What are the primary functions of CD81 in human cells?

CD81 serves multiple critical biological functions:

CD81 deficiency in humans results in immunological abnormalities, particularly affecting B cell function, demonstrating its non-redundant roles in the immune system .

What expression systems are most effective for recombinant production of human CD81?

Pichia pastoris has proven to be an exceptionally effective expression system for recombinant human CD81 production, yielding approximately 1.75 mg/L of culture of functional protein . This methylotrophic yeast provides significant advantages over prokaryotic systems like E. coli, including eukaryote-like post-translational modifications and proper protein folding. For optimal expression, researchers should consider:

• Removing palmitoylation sites (Cys to Ala mutations) in the central segment of hCD81

• Introducing a C-terminal His6 tag for purification

• Optimizing the coding sequence with appropriate Kozak consensus sequence

• Using immunoblotting and immunofluorescence with conformationally-specific antibodies to verify proper folding

• Performing circular dichroism analysis to confirm α-helical secondary structure

This approach produces highly-pure, homogeneous, active human CD81 that maintains its native conformation and functional properties, as demonstrated by its ability to interact with Hepatitis C virus E2 glycoprotein .

What techniques are most effective for studying CD81 protein-protein interactions?

Multiple complementary techniques provide comprehensive insights into CD81's interaction network:

Combining these approaches is particularly powerful, as demonstrated in studies that identified specific regions of CD81 required for interaction with CD44 and subsequent effects on cellular functions like mammosphere formation .

How does CD81 regulate B cell development and function?

CD81 plays a critical role in B cell biology through its relationship with CD19:

• CD19 Complex Formation: CD81 is an essential component of the CD19-CD21-CD81 signaling complex on B cells, which functions as a co-receptor for the B cell antigen receptor (BCR) .

• CD19 Membrane Expression: CD19 membrane expression critically depends on CD81. In CD81-deficient patients, CD19 expression on B cells is dramatically reduced, as demonstrated through retroviral transduction experiments .

• B Cell Signaling: The intact CD19 complex lowers the threshold for B cell activation by bridging innate and adaptive immune responses. B cells from CD81-deficient patients show impaired activation upon stimulation via the BCR, similar to CD19-deficient patients .

• Clinical Consequences: CD81 gene defects in humans result in antibody deficiency syndrome with hypogammaglobulinemia and increased susceptibility to infections, highlighting its non-redundant function in human humoral immunity .

This intimate relationship makes CD81 essential for proper B cell function, with its absence leading to significant immunological defects .

How does CD81 contribute to T cell activation and cytokine production?

CD81 makes significant contributions to T cell activation through several mechanisms:

This dual effect explains the seemingly contradictory observations of general T cell costimulation versus specific Th2 biasing by CD81 in human T cells .

What are the immunological consequences of CD81 gene mutations in humans?

CD81 mutations can profoundly affect immune function, primarily impacting B cell function and antibody production:

These findings establish CD81 deficiency as a distinct cause of antibody deficiency syndrome, highlighting the non-redundant functions of CD81 in human B cell function .

How does CD81 contribute to triple-negative breast cancer progression?

CD81 plays multiple roles in triple-negative breast cancer (TNBC) progression:

• Promotion of Tumor Self-Renewal: CD81 is critical for mammosphere formation, a measure of self-renewal capacity. CD81 knockout (CD81KO) in TNBC cells significantly reduces mammosphere formation beyond minor alterations in cell growth .

• Interaction with CD44: CD81 directly interacts with CD44, a known promoter of tumor initiation and metastasis. This interaction occurs through specific regions, as CD81 mutants that cannot bind CD44 show compromised mammosphere formation capacity .

• Regulation of Downstream Pathways: Phosphoproteomic profiling revealed that CD81 regulates endocytosis pathways, which was unexpected. This regulation is coordinated with CD44 and coupled with metabolic regulation, potentially providing energy for cancer progression .

• Metastasis Promotion: CD81 enhances tumor clustering and lung metastasis in TNBC. In patient-derived xenograft models, CD81+ tumor cells showed enhanced tumorigenicity compared to CD81- cells .

These findings highlight CD81 as a potential therapeutic target in TNBC, particularly through its role in promoting self-renewal and interaction with CD44 .

What is the role of CD81 in pathogen invasion, particularly with Salmonella enterica?

CD81 facilitates Salmonella enterica invasion into epithelial host cells through several mechanisms:

• Enhancement of Bacterial Entry: Overexpression of CD81 in HepG2 cells enhances invasion of various Salmonella serovars, while deletion of CD81 by CRISPR/Cas9 in intestinal epithelial cells reduces S. Typhimurium invasion .

• Species-Specific Effect: The facilitation of Salmonella invasion is species-specific, with only human CD81 (not rat CD81) enhancing invasion, suggesting evolutionary adaptation of the pathogen to its human host .

• Invasion Site Recruitment: CD81 is recruited specifically to the entry site of S. Typhimurium during invasion, but not during adhesion to the host cell surface, indicating a role in the invasion process rather than initial bacterial attachment .

• Tetraspanin Cooperation: Another tetraspanin, CD9, is also recruited to Salmonella entry sites, suggesting cooperation between different tetraspanin family members during pathogen invasion .

Understanding CD81's role in bacterial invasion provides insights into host-pathogen interactions and may offer potential targets for therapeutic intervention against Salmonella infections .

How does CD81 interact with CD44 to regulate cellular functions?

CD81 and CD44 interact to regulate multiple cellular functions through specific molecular mechanisms:

These findings highlight the complex interplay between CD81 and CD44 in regulating cellular functions, particularly in cancer cell self-renewal .

What are the implications of CD81 palmitoylation on its function?

Palmitoylation of CD81 has significant implications for its structure, localization, and function:

When studying CD81, researchers should consider how palmitoylation status might influence the protein's behavior and whether experimental modifications to these sites could affect the interpretation of functional data .

What are the current gaps in our understanding of human CD81?

Despite significant advances in CD81 research, several important knowledge gaps remain. The detailed structure of full-length CD81, particularly the positioning of EC1 and the transmembrane domains, is still not completely understood . The precise molecular mechanisms through which CD81 regulates endocytosis and how this connects to its various cellular functions needs further investigation . Additionally, the apparent redundancy of CD81 in T cells (as evidenced by lack of T cell defects in CD81-deficient patients) versus its non-redundant role in B cells requires further exploration .