CD320 Human
Description
Molecular Structure of CD320
CD320 is a transmembrane protein with distinct structural domains essential for its function:
The crystal structure of CD320 in complex with holo-TC (2.1 Šresolution) reveals a 1:1 binding stoichiometry. The interface involves ~900 Ų buried surface area, with LDLR-A2 contributing 486 Ų and LDLR-A1 contributing 405 Ų . Key interactions include:
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Ca²⁺ coordination: Acidic residues (e.g., Asp153, Asp157) in LDLR-A2 stabilize binding .
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pH sensitivity: His104 (TC) and His155 (CD320) form a "histidine switch" that destabilizes the complex at low pH (e.g., in endosomes) .
Functional Characteristics
CD320 is ubiquitously expressed but shows elevated levels in proliferating cells, including cancer cells and germinal center B-cells .
Genetic Mutations
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CD320ΔE88: A disease-causing mutant with preserved TC binding but altered stability .
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Methylmalonic aciduria: Linked to CD320 mutations that impair Cbl transport .
Autoimmune Disorders
Recent studies identify anti-CD320 autoantibodies in:
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Autoimmune B₁₂ central deficiency: Causes neurological deficits (e.g., ataxia, cognitive decline) by blocking Cbl transport across the blood-brain barrier .
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Systemic lupus erythematosus (SLE): 21.4% of neuropsychiatric SLE patients show anti-CD320 seropositivity .
Product Specs
Cluster of Differentiation 320 (CD320), a member of the low-density lipoprotein receptor family, plays a crucial role in cobalamin (vitamin B12) uptake. It achieves this by binding to the transcobalamin II (TCN2)-cobalamin complex and facilitating its internalization through endocytosis. CD320 is characterized by two low-density lipoprotein receptor type A domains separated by a complement-like cysteine-rich region. Beyond its role in cobalamin uptake, CD320 also functions as a costimulator for B cell and follicular dendritic cells, contributing to their activation and response. Additionally, CD320 has been observed to enhance the proliferation of plasma cell (PC) precursors induced by Interleukin-10 (IL-10).
Recombinant human CD320, expressed in Sf9 insect cells using a baculovirus expression system, is a single, glycosylated polypeptide chain. It consists of 438 amino acids (residues 31-229a.a.), resulting in a molecular weight of 47.4 kDa. However, on SDS-PAGE, it appears as a band at approximately 57-70 kDa due to glycosylation. The protein is engineered with a 239 amino acid human IgG-His tag at the C-terminus to facilitate purification, which is achieved through proprietary chromatographic techniques.
The CD320 protein is supplied as a 0.5 mg/mL solution in Phosphate Buffered Saline (pH 7.4) containing 10% glycerol.
For short-term storage (2-4 weeks), the protein can be stored at 4°C. For long-term storage, it is recommended to store the protein at -20°C. The addition of a carrier protein such as HSA or BSA (0.1%) is recommended for long-term storage. Avoid repeated freeze-thaw cycles to maintain protein stability.
The purity of the CD320 protein is greater than 90% as determined by SDS-PAGE analysis.
CD320 antigen isoform 1, CD320, 8D6, 8D6A, TCBLR, FDC-SM-8D6, FDC-signaling molecule 8D6.
Sf9, Baculovirus cells.
LEAAASPLST PTSAQAAGPS SGSCPPTKFQ CRTSGLCVPL TWRCDRDLDC SDGSDEEECR IEPCTQKGQC PPPPGLPCPC TGVSDCSGGT DKKLRNCSRL ACLAGELRCT LSDDCIPLTW RCDGHPDCPD SSDELGCGTN EILPEGDATT MGPPVTLESV TSLRNATTMG PPVTLESVPS VGNATSSSAG DQSGSPTAYL EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKHHHHHH.
Q&A
What is the structural basis of transcobalamin recognition by human CD320?
The crystal structure of human holo-TC in complex with the extracellular domain of CD320 provides visualization of this critical interaction . CD320 features an N-terminal extracellular fragment comprising two LDLR type A (LDLR-A) domains separated by a linker region. The observed interaction chemistry explains both CD320's high affinity for transcobalamin and its lack of haptocorrin binding. For experimental studies, researchers should consider that this structural understanding provides the foundation for interpreting how mutations might affect transcobalamin recognition and subsequent cobalamin uptake mechanisms.
How does CD320 participate in cellular vitamin B12 uptake?
CD320 functions as a ubiquitous cell surface receptor that captures transcobalamin (TC) from plasma . This initiates receptor-mediated endocytosis through a Ca²⁺-dependent complex formation between TC and CD320. Following internalization, the TC-cobalamin complex undergoes processing in the endosomal/lysosomal system, allowing vitamin release for cellular utilization. Research methodologies focusing on this pathway should account for the fact that defects anywhere in this uptake system can lead to cobalamin deficiency and potentially severe developmental consequences .
What is the functional significance of different CD320 domains?
CD320, as a member of the Low-density Lipoprotein Receptor (LDLR) family, contains two LDLR type A domains (LDLR-A1 and LDLR-A2) in its extracellular portion . Experimental evidence demonstrates that stable complexes with TC form with both wild-type CD320 and the isolated LDLR-A2 domain , indicating the crucial role of LDLR-A2 in TC binding. When designing domain-specific experiments, researchers should note that these LDLR-A domains contain conserved calcium-binding sites essential for proper receptor folding and function in capturing TC.
How does pH affect CD320-TC complex stability during endocytosis?
Researchers have demonstrated significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release mechanism during endocytosis . This pH sensitivity represents a critical aspect of receptor-mediated endocytosis, allowing TC-CD320 complex formation at neutral plasma pH with subsequent dissociation in the increasingly acidic endosomal environment. Experimental designs investigating this phenomenon should include binding studies across pH ranges (7.4-5.0) to model conditions throughout the endocytic pathway, providing insights into how mutations might affect this pH-dependent interaction.
What is the prevalence of CD320 p.E88del across different populations?
Population frequency data reveals significant ethnic variation in CD320 p.E88del prevalence. According to gnomAD data, deletion allele frequencies range from 0.003 in African and South Asian populations to 0.014 in Latino and East Asian populations . The table below summarizes these findings:
| Population | CD320 p.E88del Frequency |
|---|---|
| European (non-Finnish) | 0.01 |
| European (Finnish) | 0.004 |
| African | 0.003 |
| Latino | 0.014 |
| East Asian | 0.014 |
| South Asian | 0.003 |
| Ashkenazi Jewish | 0.01 |
New York State data confirms these patterns, with approximately 1 homozygote expected per 10,000 individuals . For population studies, researchers should ensure adequate sample sizes and ethnic diversity to accurately capture variant frequencies.
What are the long-term clinical outcomes for individuals with CD320 p.E88del homozygosity?
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Language acquisition deficit
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Working memory deficits
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ADHD
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Autism spectrum disorder
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Anxiety
The remaining individuals developed normally without apparent neurological issues . This heterogeneity suggests that additional genetic or environmental factors likely modify the phenotypic expression of CD320 mutations. Research methodologies should incorporate comprehensive neurodevelopmental assessments alongside biochemical markers when studying long-term outcomes.
How should treatment approaches be evaluated for individuals with CD320 p.E88del homozygosity?
Based on case studies, various treatment approaches have been employed for CD320 p.E88del homozygotes, including:
| Treatment Approach | Implementation | Duration | Outcome |
|---|---|---|---|
| IM hydroxocobalamin | Daily to 3x weekly | 6 weeks to ongoing | Normal MMA/homocysteine |
| Oral cyanocobalamin | Daily (50-1000 μg) | Variable | Normal biochemical parameters |
| Combination therapy | With carnitine, folate, betaine | Variable | Normal biochemical parameters |
Notably, 6 of 9 treated cases discontinued therapy before one year of age with continued normal biochemical parameters . This suggests that randomized controlled trials comparing different intervention strategies (including no treatment) are needed to determine optimal management. Research methodologies should include standardized outcome measures across biochemical, growth, and neurodevelopmental domains.
What biochemical markers should be monitored when studying CD320 function?
When investigating CD320 function, researchers should employ a comprehensive panel of biomarkers:
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Propionylcarnitine (C3) and C3:C2 ratio - elevated in newborn screening of affected individuals
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Methylmalonic acid (MMA) - in plasma and urine
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Total homocysteine (tHcy) - reflects methionine synthase activity
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Total circulating cobalamin - typically elevated in CD320 mutations due to reduced cellular uptake
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Holotranscobalamin (holoTC) - elevated circulating levels in individuals with CD320 mutations
These markers should be assessed both at baseline and in response to interventions. Studies should incorporate age-appropriate reference ranges as most CD320 p.E88del homozygotes show normalization of these markers after the newborn period .
What methods are appropriate for quantifying TC-CD320 interactions in vitro?
Based on published methodologies, researchers investigating TC-CD320 interactions should consider:
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Solid-phase binding assays - for quantitating in vitro affinity of TC-CD320 complexes
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Thermostability analysis - to assess complex stability under various conditions
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Crystallographic studies - for detailed visualization of interaction interfaces
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Surface plasmon resonance - to measure real-time binding kinetics
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pH-dependent binding assays - to recapitulate endosomal conditions
Experiments should include both wild-type and mutant proteins (particularly CD320ΔE88) to assess the effects of specific amino acid changes on binding properties. Additionally, testing should occur across pH ranges to model physiological conditions during endocytosis.
What cell models best represent CD320-mediated cobalamin uptake?
When selecting cellular models for CD320 research, investigators should consider:
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Primary human fibroblasts - particularly useful when derived from individuals with known CD320 genotypes
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HepG2 cells - a liver cell line expressing endogenous CD320
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Engineered cell lines with CD320 knockout/knockin - allowing precise genotype-phenotype studies
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Differentiated neural cells - to study CD320 function in neurological development
Experimental designs should include appropriate controls:
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Positive controls: cells expressing wild-type CD320
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Negative controls: CD320 knockout cells
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CD320 mutant cells (e.g., p.E88del homozygous)
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Rescue controls: mutant cells with reintroduced wild-type CD320
These models enable investigation of CD320-mediated cobalamin uptake under controlled conditions, allowing for precise quantification of functional consequences resulting from specific genetic variants.
Product Science Overview
CD320 is a heavily glycosylated protein that is expressed as a noncovalent dimer. It consists of a 199 amino acid extracellular domain with two cysteine-rich LDLR-A repeats, a 21 amino acid transmembrane segment, and a 32 amino acid cytoplasmic domain . Within the extracellular domain, human CD320 shares 52% amino acid sequence identity with mouse and rat CD320 .
The primary function of CD320 is to mediate the uptake of transcobalamin-bound cobalamin into cells. This process is essential for the proper functioning of various biological processes, including DNA synthesis and red blood cell formation.
Recombinant human CD320 is produced using a mouse myeloma cell line (NS0) and is available in various formulations for research purposes. The recombinant protein is typically purified to a high degree of purity (>90%) and is used in various applications, including functional assays and binding studies .
The recombinant human CD320 protein is often used in studies to understand its role in cobalamin metabolism and its potential implications in diseases related to vitamin B12 deficiency. It is also used in the development of therapeutic interventions targeting cobalamin uptake pathways.
Recombinant human CD320 is utilized in several research areas, including:
- Functional ELISA: To measure its binding ability to transcobalamin.
- Cell Culture: To study its role in cobalamin uptake and metabolism.
- Therapeutic Research: To develop potential treatments for conditions related to vitamin B12 deficiency.
