Recombinant Human Putative cationic amino acid transporter 3-like protein

What is the human cationic amino acid transporter 3 (hCAT-3) and how does it function?

Human cationic amino acid transporter 3 (hCAT-3) is a member of the system y+ family of transporters, characterized by sodium-independent transport of cationic amino acids. It is encoded by the SLC7A3 gene and functions as a selective transporter for cationic L-amino acids located in the plasma membrane . Unlike its rodent counterparts, human CAT-3 exhibits a maximal transport activity similar to other CAT proteins and demonstrates high selectivity for cationic amino acids . The transporter plays a critical role in cellular uptake of essential amino acids such as arginine and lysine, which are necessary for various cellular processes including protein synthesis and cell signaling pathways.

How does hCAT-3 differ from other members of the CAT family?

hCAT-3 belongs to a family of at least five distinct carrier proteins forming the mammalian cationic amino acid transporters (CATs). While it shares functional similarities with other CAT proteins, hCAT-3's apparent substrate affinity and sensitivity to trans-stimulation most closely resembles hCAT-2B . This distinguishes it from CAT-1, which has different kinetic properties. One significant difference between hCAT-3 and other family members lies in its tissue distribution pattern, with hCAT-3 showing notably high expression in the thymus and other peripheral tissues, contrasting with the brain-specific expression of rodent CAT-3 .

What is the expression pattern of hCAT-3 in human tissues?

Unlike rodent CAT-3, which is expressed exclusively in central neurons in adult rats and mice, human CAT-3 expression is not restricted to the brain. Research has shown that by far the highest expression of hCAT-3 is found in the thymus . Other peripheral tissues show equal or higher expression levels than most brain regions, indicating that hCAT-3 is not a neuron-specific transporter in humans . This contrasting expression pattern between human and rodent CAT-3 suggests potentially different physiological roles across species and highlights the importance of studying the human transporter specifically rather than extrapolating from rodent models.

What are the structural characteristics of hCAT-3?

Human CAT-3 is a glycosylated plasma membrane protein with multiple transmembrane domains, characteristic of amino acid transporters . While the search results don't provide the exact number of transmembrane domains for hCAT-3 specifically, related amino acid transporters typically contain 9-14 transmembrane domains. The protein is encoded by the SLC7A3 gene located on the X chromosome (as indicated by its OMIM number: 300443) . Post-translational modifications, particularly glycosylation, are important for proper folding, trafficking, and function of the transporter at the plasma membrane.

What is the substrate specificity of hCAT-3?

hCAT-3 is highly selective for cationic L-amino acids, primarily transporting arginine, lysine, and ornithine . This selectivity is characteristic of the system y+ family of transporters to which it belongs. Unlike rat and murine CAT-3 proteins, which have been reported to be inhibited by neutral and anionic L-amino acids as well as D-arginine, human CAT-3 appears to maintain stricter substrate selectivity . Its transport mechanism is sodium-independent, another defining feature of the system y+ transporters .

What are the kinetic properties of hCAT-3 transport?

Transport studies in Xenopus laevis oocytes have revealed that hCAT-3 demonstrates kinetic properties similar to other CAT proteins, particularly hCAT-2B . The transport mechanism shows Michaelis-Menten kinetics, indicative of a carrier-mediated transport process. While the exact Km values for hCAT-3 aren't provided in the search results, the affinity for substrates and sensitivity to trans-stimulation are noted to be similar to hCAT-2B. In amino acid transporters, trans-stimulation refers to the phenomenon where the presence of transportable substrates on one side of the membrane increases the transport rate of substrates on the opposite side, a characteristic that appears to be present in hCAT-3 function .

What expression systems are commonly used to study hCAT-3 function?

The Xenopus laevis oocyte expression system has been successfully used to study the transport function of hCAT-3 . This system involves:

• Cloning the complete coding region of human CAT-3

• Synthesizing mRNA from the cloned cDNA

• Injecting the synthetic mRNA into Xenopus laevis oocytes

• Allowing protein expression for 3 days at 18°C

• Performing functional analyses in groups of 5-10 oocytes per assay

This heterologous expression system provides a clean background for functional studies, as oocytes generally have low endogenous amino acid transport activities. Mammalian cell lines such as HEK 293 cells have also been used to study cationic amino acid transporters, though specific examples for hCAT-3 aren't detailed in the search results .

How can transport activity of recombinant hCAT-3 be measured experimentally?

Transport activity can be measured using radiolabeled amino acids as tracers. A typical experimental protocol includes:

• Rinsing the oocytes expressing hCAT-3 briefly in uptake buffer

• Transferring oocytes to a culture dish containing uptake buffer

• Incubating oocytes with 50 μM L-amino acids plus corresponding [³H]-labeled L-amino acids as tracers for 15 or 30 minutes

• Washing the oocytes four times in uptake buffer

• Lysing the oocytes in 2% SDS

• Measuring the accumulated radioactivity with a scintillation counter

For specificity studies, competition assays can be performed by measuring labeled amino acid uptake in the presence of excess (e.g., 5 mM) non-radioactive amino acids. Na⁺-dependence can be investigated by substituting Na⁺ with choline⁺ or Li⁺ in the uptake buffer .

What controls should be included when studying recombinant hCAT-3 transport properties?

When studying recombinant hCAT-3, several controls should be included:

• Negative controls: Oocytes injected with DEPC-H₂O instead of mRNA to establish baseline transport levels

• Substrate specificity controls: Including various amino acids (cationic, neutral, and anionic) to confirm selectivity

• Ion dependency controls: Testing transport in Na⁺-free conditions to confirm Na⁺-independent transport characteristic of system y⁺ transporters

• Time-course measurements: To establish linear range of uptake

• Concentration-dependent measurements: To determine kinetic parameters

• Trans-stimulation studies: To examine the effect of internal substrates on transport rates

These controls help verify that the observed transport activity is indeed mediated by the recombinant hCAT-3 and characterize its functional properties.

How can researchers investigate the regulation of hCAT-3 activity?

Regulation of hCAT-3 activity can be studied at multiple levels:

• Transcriptional regulation: Analysis of promoter activity and regulatory elements in the SLC7A3 gene

• Post-translational modifications: Investigation of glycosylation and phosphorylation states and their impact on transport activity

• Trafficking regulation: Studies on internalization and recycling mechanisms, similar to those described for hCAT-1 where protein kinase C activation promotes internalization

• Substrate-induced regulation: Examination of trans-stimulation effects where intracellular substrates influence transport rates

• Physiological regulators: Investigation of hormones, cytokines, or growth factors that might regulate transporter expression or activity

Methodologies may include site-directed mutagenesis to identify regulatory domains, inhibitor studies to examine signaling pathways involved in regulation, and imaging techniques to study trafficking dynamics.

What are the implications of hCAT-3's distinct expression pattern compared to rodent CAT-3?

The distinct expression pattern of hCAT-3 compared to rodent CAT-3 has several research implications:

• Physiological role: The high expression in thymus suggests a possible role in immune function not present in rodent models

• Animal model limitations: Rodent models may not accurately represent human CAT-3 function in peripheral tissues

• Tissue-specific regulation: Different regulatory mechanisms may control hCAT-3 expression across various tissues

• Evolutionary divergence: The species differences suggest evolutionary divergence in function

• Therapeutic targeting: Different targeting strategies may be needed for human conditions compared to those developed in rodent models

Researchers should be cautious about extrapolating findings from rodent CAT-3 studies to human conditions, particularly for peripheral tissues where expression patterns differ significantly .

How might hCAT-3 function be studied in the context of specific physiological or pathological conditions?

To study hCAT-3 function in specific physiological or pathological contexts, researchers could:

• Generate tissue-specific or inducible knockout/knockdown models: Using CRISPR-Cas9 or RNAi technologies in relevant cell lines

• Develop specific inhibitors or activators: To modulate transporter function

• Study disease models: Investigate alterations in hCAT-3 expression or function in conditions like immune disorders (given its high expression in thymus)

• Nutrient sensing pathways: Explore the role of hCAT-3 in cellular responses to amino acid availability

• Metabolic flux analysis: Examine how hCAT-3 influences cellular metabolism through regulation of cationic amino acid availability

For example, researchers studying immune cell function might investigate how hCAT-3-mediated arginine transport affects T-cell proliferation or cytokine production in the thymus, where hCAT-3 shows particularly high expression .

What are important considerations when working with recombinant hCAT-3 protein?

When working with recombinant hCAT-3 protein, researchers should consider:

• Expression system: Different expression systems (bacterial, insect, mammalian) may affect protein folding, post-translational modifications, and functionality

• Purification strategy: Membrane proteins require specialized purification techniques using detergents

• Protein solubility: As a membrane protein, maintaining proper folding in solution requires appropriate detergents or reconstitution into lipid environments

• Storage conditions: Typically requiring glycerol and specific buffer compositions to maintain stability

• Functional verification: Confirming that the recombinant protein retains transport activity

• Presence of tags: Considering the potential impact of affinity tags on function and developing tag-removal strategies if necessary

Commercially available recombinant hCAT-3 proteins may already address some of these considerations in their formulation and storage recommendations.

What methods can be used to verify the quality and functionality of recombinant hCAT-3?

Several analytical methods can verify recombinant hCAT-3 quality and functionality:

• SDS-PAGE and Western blotting: To confirm protein size, purity, and identity

• Glycosylation analysis: To verify proper post-translational modifications

• Circular dichroism: To assess secondary structure

• Reconstitution into liposomes: For functional studies outside of cellular contexts

• Transport assays: Using radioactive or fluorescently labeled substrates to confirm functionality

• Thermal stability assays: To assess protein stability

• Surface plasmon resonance or microscale thermophoresis: To study substrate binding kinetics

A combination of these approaches provides comprehensive quality assessment of recombinant hCAT-3 preparations.

How can recombinant hCAT-3 be incorporated into different experimental platforms?

Recombinant hCAT-3 can be incorporated into various experimental platforms:

Each platform offers distinct advantages and limitations, and selection should be based on specific research questions and available resources.

How does hCAT-3 compare functionally to other human cationic amino acid transporters?

The human cationic amino acid transporter family includes multiple members with distinctive properties:

hCAT-3's substrate affinity and sensitivity to trans-stimulation most closely resembles hCAT-2B, though their tissue distribution patterns differ significantly .

What evolutionary insights can be gained from studying species differences in CAT-3?

The significant differences between human and rodent CAT-3 in terms of tissue expression patterns and functional characteristics provide valuable evolutionary insights:

• Functional divergence: Despite sequence homology, human and rodent CAT-3 have evolved different tissue specificities and possibly different physiological roles

• Selective pressures: Different evolutionary pressures may have shaped CAT-3 function across species, possibly related to dietary adaptations or immune system requirements

• Regulatory evolution: The divergent expression patterns suggest evolution of different regulatory mechanisms controlling gene expression

• Functional redundancy: In tissues where rodent CAT-3 is absent but human CAT-3 is present, other transporters may compensate in rodents

• Translational implications: These differences highlight the importance of studying human proteins directly rather than relying solely on rodent models

The high expression of hCAT-3 in thymus compared to its neuron-specific expression in rodents suggests a potential evolutionary adaptation related to immune function in humans .

What are promising research directions for understanding hCAT-3 function in human physiology?

Several promising research directions for hCAT-3 include:

• Immune function: Investigating the role of hCAT-3 in T-cell development and function given its high expression in thymus

• Brain function: Comparing the neuronal roles of hCAT-3 to those of rodent CAT-3 despite lower expression levels

• Tissue-specific regulation: Elucidating the mechanisms controlling differential expression across tissues

• Metabolic integration: Understanding how hCAT-3-mediated amino acid transport integrates with cellular metabolic networks

• Disease associations: Exploring potential links between hCAT-3 variants or expression changes and human diseases

• Therapeutic targeting: Developing selective modulators of hCAT-3 function for possible therapeutic applications

These directions could significantly advance our understanding of cationic amino acid transport in human physiology and pathology.

What methodological advances would benefit research on hCAT-3 and related transporters?

Several methodological advances would benefit hCAT-3 research:

• Development of selective inhibitors: Currently lacking for specific CAT family members

• Improved structural characterization: Cryo-EM or crystallography studies of human CAT proteins

• Advanced imaging techniques: Methods for real-time visualization of transport activity in living cells

• Tissue-specific conditional knockout models: For studying physiological roles in specific contexts

• Single-cell analysis methods: To understand heterogeneity of expression and function

• Biosensor development: Genetically encoded sensors for monitoring substrate transport

• Systems biology approaches: Integration of transport data with metabolomic and proteomic analyses

These advances would provide powerful new tools for understanding the functional significance of hCAT-3 in human physiology and disease.