Recombinant Macaca mulatta Leukocyte-specific transcript 1 protein (LST1)

What is LST1 and what is its basic structure in Macaca mulatta?

LST1 is a small transmembrane adaptor protein approximately 97 amino acids long. The protein contains a short extracellular segment with a dimerization cysteine, a single transmembrane domain followed by a palmitoylation site, and a cytoplasmic tail containing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). While human LST1 has at least 16 splice variants, evidence suggests that rhesus macaques may have a more limited number of splice variants, similar to mice which have only two identified RNA splice forms .

Where is LST1 typically expressed in rhesus macaques?

Based on comparative studies with other species, LST1 in rhesus macaques is primarily expressed in cells of the myeloid lineage, including macrophages, dendritic cells, monocytes, and granulocytes. This expression pattern appears to be conserved across species, as demonstrated in studies using monoclonal antibodies against LST1 in various organisms . Tissue distribution likely includes lymphoid organs and sites of inflammation, though species-specific variation may exist.

What are the common methods for detecting Macaca mulatta LST1 expression?

Detection of LST1 in rhesus macaques can be accomplished through:

• Western blotting using antibodies specific to macaque LST1

• Immunoprecipitation with monoclonal antibodies developed against conserved epitopes

• RT-PCR for transcript analysis (noting that splice variation may be less extensive than in humans)

• Flow cytometry for cell-type specific expression profiling

Monoclonal antibodies developed against specific domains of LST1, such as the intracellular domain beginning at Cys40, have proven effective in detecting LST1 in immunological studies .

How does recombinant Macaca mulatta LST1 differ from human LST1 in terms of structure and function?

While the core functional domains of LST1 are conserved between humans and rhesus macaques, significant differences exist in their splicing patterns. Human LST1 exhibits extensive alternative splicing with 16 documented variants, whereas evidence from comparative studies with mice suggests that rhesus macaques may express fewer splice variants . These structural differences likely translate to species-specific functional variations, particularly in immune regulation pathways.

What is the recommended approach for producing functionally active recombinant Macaca mulatta LST1?

For optimal production of functionally active recombinant Macaca mulatta LST1:

• Expression System Selection: Mammalian expression systems (HEK293 or CHO cells) are preferred over bacterial systems for proper post-translational modifications, particularly palmitoylation.

• Construct Design:

  • Include a cleavable tag (His6 or FLAG) for purification

  • Consider expressing the intracellular domain separately (starting at Cys40) for antibody production or interaction studies

  • Ensure proper codon optimization for the expression system

• Purification Strategy:

  • Detergent solubilization with mild non-ionic detergents (e.g., digitonin or DDM)

  • Two-step purification using affinity chromatography followed by size exclusion

• Functional Verification:

  • Assess membrane integration using liposome reconstitution assays

  • Verify phosphorylation states of ITIMs under different conditions

  • Evaluate binding to known interaction partners (SHP-1, SHP-2, IκBα)

What are the key experimental considerations when studying LST1's role in inflammatory responses in Macaca mulatta models?

When investigating LST1's role in inflammatory responses in rhesus macaque models:

• Baseline Characterization:

  • Document tissue-specific expression levels across healthy macaque tissues

  • Establish leukocyte subset composition in relevant tissues (lymph nodes, spleen, peripheral blood)

• Inflammatory Challenge Models:

  • Consider dextran sodium sulfate (DSS) induced colitis, which has revealed significant phenotypes in LST1-deficient mice

  • Viral infection models may be particularly relevant, as LST1 expression increases under these conditions

  • LPS or TNFα challenge for acute inflammation assessment

• Measurement Parameters:

  • Monitor dynamic changes in LST1 expression during inflammatory progression

  • Assess alterations in leukocyte subset composition using flow cytometry panels including markers for:

    • T cells (CD4+, CD8+)

    • B cells (CD19+, MHCII+)

    • NK cells (CD5-, CD161+)

    • Monocytes (CD11b+, Ly6C+, Ly6G-)

    • Neutrophils (CD11b+, Ly6C med, Ly6G+)

    • Macrophages (CD11b+, F4/80+)

    • Dendritic cells (CD11b+/-, MHCII+, CD11c+)

• Signaling Pathway Analysis:

  • Examine NF-κB pathway components, particularly IκBα levels

  • Assess SHP-1 and SHP-2 recruitment and phosphorylation states

What sequencing methodologies are recommended for characterizing Macaca mulatta LST1 variants?

Single-molecule real-time (SMRT) sequencing using the Pacific Biosciences Sequel platform is recommended for comprehensive characterization of LST1 transcripts in rhesus macaques. This approach has been successfully employed for other immune-related gene clusters in macaques, such as KIR genes . SMRT sequencing offers several advantages:

• Long read lengths capable of spanning entire LST1 transcripts

• Direct detection of splice variants without assembly bias

• Identification of novel alleles and extension of previously reported sequences

• Ability to determine haplotype structures

This approach is superior to conventional methods like Sanger sequencing or Roche/454 pyrosequencing, which often fail to define complete transcripts or achieve allele-level resolution .

How can researchers generate specific antibodies against Macaca mulatta LST1 for immunological studies?

Generation of specific antibodies against Macaca mulatta LST1 requires:

• Antigen Preparation:

  • Recombinant expression of the intracellular domain (starting at Cys40) in E. coli has proven successful

  • Alternatively, consider synthesizing unique peptide sequences specific to macaque LST1

• Immunization Strategy:

  • For monoclonal antibodies, immunize LST1-deficient mice with the recombinant protein to maximize immune response

  • For polyclonal antibodies, rabbits or goats may be used with purified protein fragments

• Antibody Screening:

  • Use both Western blot and immunoprecipitation assays for validation

  • Confirm specificity by testing against lysates from LST1-knockout cells

  • Evaluate cross-reactivity with human LST1 to determine conservation of epitopes

• Purification and Characterization:

  • After hybridoma generation, clone antibody-producing cells by limiting dilution

  • Characterize antibodies for application-specific performance (WB, IP, FACS, IHC)

What approaches can be used to study the functional impact of LST1 in Macaca mulatta immune response?

To investigate LST1's functional impact on rhesus macaque immune responses:

• Gene Modification Approaches:

  • CRISPR/Cas9-mediated knockout in macaque cell lines

  • RNAi-based knockdown for transient suppression

  • Overexpression systems with wild-type or mutant variants

• Ex Vivo Systems:

  • Primary macaque peripheral blood mononuclear cell (PBMC) cultures

  • Macrophage differentiation and activation assays

  • Dendritic cell maturation and antigen presentation studies

• In Vivo Analysis:

  • Longitudinal monitoring of leukocyte subset composition in relevant tissues

  • Challenge studies with inflammatory stimuli (viral, bacterial, chemical)

  • Comparative analysis between wild-type and LST1-modified systems

• Molecular Interaction Studies:

  • Co-immunoprecipitation to identify binding partners

  • Phosphorylation analysis following immune stimulation

  • Evaluation of SHP-1/SHP-2 recruitment to ITIMs

How does LST1 function compare between Macaca mulatta and other primate models?

Comparative analysis of LST1 between rhesus macaques and other primates reveals both conservation and divergence:

• Structural Comparison:

  • Humans exhibit extensive alternative splicing (16+ variants) while non-human primates likely have fewer splice variants, similar to mice (2 variants)

  • Core functional domains (transmembrane region, ITIMs) show high conservation across primates

  • Species-specific differences may exist in the extracellular domain

• Expression Patterns:

  • Myeloid lineage-specific expression appears conserved across primates

  • Regulatory elements controlling expression under inflammatory conditions may show species-specific variations

  • Response to specific pathogens likely reflects evolutionary adaptation

• Functional Conservation:

  • Immune regulatory functions through ITIM domains likely conserved

  • Formation of tunneling nanotubes, as observed in human cells, may be present in macaque cells

  • Species-specific differences in inflammatory response modulation reflect evolutionary pressures

• Evolutionary Context:

  • LST1 location near the MHCIII locus suggests conserved genomic organization across primates

  • Selective pressures from species-specific pathogens may drive functional divergence

What are the key differences in experimental design when studying LST1 in Macaca mulatta compared to murine models?

When transitioning from murine to Macaca mulatta models for LST1 research, several key experimental design considerations must be addressed:

What genomic techniques are most effective for studying LST1 gene structure and regulation in Macaca mulatta?

For comprehensive analysis of LST1 gene structure and regulation in rhesus macaques:

• Whole Genome Sequencing:

  • Long-read sequencing technologies (PacBio, Oxford Nanopore) provide superior resolution of complex genomic regions

  • Short-read approaches (Illumina) with specialized assembly algorithms for complementary coverage

• Transcriptome Analysis:

  • SMRT sequencing for full-length transcript characterization

  • RNA-seq for quantitative expression analysis across tissues and conditions

  • Single-cell RNA-seq to resolve cell type-specific expression patterns

• Chromatin Structure and Regulation:

  • ATAC-seq to identify open chromatin regions around the LST1 locus

  • ChIP-seq for histone modifications and transcription factor binding

  • HiC or Capture-C for three-dimensional chromatin interactions

• Promoter and Enhancer Analysis:

  • Reporter assays to characterize regulatory elements

  • CRISPR interference/activation to validate enhancer function

  • Comparative genomics to identify conserved regulatory regions across primates

What are common challenges in expressing recombinant Macaca mulatta LST1 and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant Macaca mulatta LST1:

• Low Expression Yields:

  • Challenge: Membrane proteins often express poorly in heterologous systems

  • Solution: Optimize codon usage for expression host; use strong inducible promoters; consider fusion partners (SUMO, MBP) to enhance solubility

• Protein Misfolding:

  • Challenge: Improper folding leading to aggregation or degradation

  • Solution: Express at lower temperatures (16-20°C); use specialized expression hosts; consider chaperone co-expression

• Post-translational Modifications:

  • Challenge: Ensuring proper palmitoylation and phosphorylation

  • Solution: Use mammalian expression systems; verify modification status by mass spectrometry

• Protein Solubilization:

  • Challenge: Membrane proteins require detergents for extraction and purification

  • Solution: Screen detergent panel (DDM, digitonin, LMNG); consider nanodiscs or SMALPs for native-like environment

• Functional Verification:

  • Challenge: Confirming biological activity of recombinant protein

  • Solution: Develop binding assays with known interaction partners; assess phosphorylation states of ITIMs

How can researchers optimize flow cytometry protocols for studying LST1 expression in Macaca mulatta leukocyte subsets?

Optimizing flow cytometry for LST1 analysis in macaque leukocytes requires:

• Antibody Selection and Validation:

  • Validate anti-LST1 antibodies specifically for macaque reactivity

  • Test multiple clones targeting different epitopes

  • Consider intracellular staining protocols for optimal detection

• Panel Design:

  • Include lineage markers specific for macaque leukocytes:

    • T cells: CD5+, CD4+/CD8+

    • B cells: CD19+, MHCII+

    • NK cells: CD5-, CD161+

    • Monocytes: CD11b+, Ly6C+, Ly6G-

    • Neutrophils: CD11b+, Ly6C med, Ly6G+

    • Macrophages: CD11b+, F4/80+

    • Dendritic cells: CD11b+/-, MHCII+, CD11c+

• Sample Preparation Optimization:

  • Fixation conditions that preserve LST1 epitopes

  • Permeabilization protocol optimization for intracellular domains

  • Red blood cell lysis methods compatible with LST1 detection

• Instrument Setup:

  • Titration of antibodies to determine optimal concentrations

  • Appropriate compensation controls for complex panels

  • Inclusion of fluorescence-minus-one (FMO) controls

• Data Analysis:

  • Sequential gating strategy for accurate subset identification

  • Consideration of LST1 expression as both percentage positive and MFI

  • Correlation of LST1 levels with activation markers