Recombinant Macaca nemestrina Ribonuclease-like protein 9 (RNASE9)

What is Ribonuclease-like protein 9 (RNASE9) and how is it classified within the ribonuclease superfamily?

RNASE9 belongs to the ribonuclease A (RNase A) superfamily, a group of secreted proteins with diverse biological functions. Despite classification in this family, RNASE9 lacks the conventional ribonucleolytic activity that characterizes many RNase A members. Studies on human RNASE9 demonstrate no detectable ribonucleolytic activity against yeast tRNA .

The RNase A superfamily traditionally functions in RNA degradation, but several members, including RNASE9, have evolved distinct functions beyond RNA catalysis. RNASE9 belongs to a subset that has lost catalytic activity but gained new functional roles in reproductive biology and host defense .

What is known about the expression pattern of RNASE9 in primates?

Based on human RNASE9 studies, this protein exhibits a highly specific expression pattern primarily in the male reproductive tract. In humans, RNASE9 expression is predominantly localized to the epididymis, with immunofluorescence analyses revealing its presence throughout this tissue .

By extension, it is reasonable to hypothesize that Macaca nemestrina RNASE9 may exhibit a similar tissue-specific expression pattern, primarily in the epididymis and potentially other regions of the male reproductive tract. In the RNase A family, tissue-specific expression patterns are often conserved across related species.

To characterize the expression pattern of RNASE9 in Macaca nemestrina, researchers should perform:

• RT-PCR analysis of RNA extracted from various tissues

• Immunohistochemistry using specific antibodies against Macaca nemestrina RNASE9

• Western blot analysis of protein extracts from different tissues

Human RNASE9 is present on the head and neck regions of ejaculated spermatozoa and in vitro capacitated spermatozoa, suggesting similar localization might be expected in Macaca nemestrina .

What is the current understanding of the physiological role of RNASE9 in primate reproductive biology?

Based on available information, particularly from human RNASE9 studies, several physiological roles have been proposed:

• Sperm Maturation: RNASE9 may contribute to biochemical modifications that spermatozoa undergo during epididymal transit. Its presence on sperm heads suggests possible involvement in membrane remodeling during maturation .

• Host Defense: Human RNASE9 exhibits antibacterial activity against E. coli, suggesting a protective role in the male reproductive tract against bacterial infections . This antimicrobial property may help maintain a sterile environment for sperm maturation and storage.

• Male Fertility: Multiple RNase family members, including RNases 9 and 10, have been implicated in sperm maturation and male fertility . RNASE9 may similarly contribute to fertility through direct interactions with spermatozoa.

• Cell Surface Interactions: The localization on sperm surfaces suggests possible roles in sperm-oocyte recognition and binding, sperm migration through the female reproductive tract, or protection from immune surveillance.

The retention of RNASE9 across primate species despite the loss of ribonucleolytic activity suggests important non-canonical functions. The evolutionary pressure to maintain this protein implies its critical role in reproductive biology.

What are the recommended methodologies for cloning and expressing recombinant Macaca nemestrina RNASE9?

Based on successful approaches used for human RNASE9 and standard recombinant protein production practices, the following comprehensive protocol is recommended:

Cloning Strategy:

• RNA Extraction and cDNA Synthesis:

  • Extract total RNA from Macaca nemestrina epididymal tissue

  • Perform reverse transcription with oligo(dT) primers using Moloney Murine Leukemia Virus reverse transcriptase at 42°C for 60 minutes

• PCR Amplification:

  • Design primers based on predicted or known Macaca nemestrina RNASE9 sequence

  • Include appropriate restriction sites (e.g., EcoRI and KpnI as used for human RNASE9)

  • Perform PCR with high-fidelity DNA polymerase

• Vector Construction:

  • For mammalian expression: Clone into pcDNA vector with a C-terminal His-tag

  • For bacterial expression: Clone into pET25b(+) vector, excluding the signal peptide sequence

Expression Systems:

Purification Methods:

• For His-tagged Proteins:

  • Use Ni-NTA affinity chromatography

  • Apply imidazole gradient elution (20-250 mM)

• For Non-tagged Proteins:

  • Use ion-exchange chromatography (DEAE sepharose)

  • Elute with NaCl gradient (0-0.5 M) in appropriate buffer

• Additional Purification Steps:

  • Size exclusion chromatography to remove aggregates

  • Endotoxin removal for immunological studies

Quality Control:

• SDS-PAGE analysis for purity assessment (>95% purity)

• Western blot for identity confirmation

• Mass spectrometry for molecular weight verification

• N-terminal sequencing to confirm correct processing

• Functional assays (e.g., antimicrobial activity testing)

This approach, adapted from methods used for human RNASE9 , provides a reliable framework for producing recombinant Macaca nemestrina RNASE9 for research purposes.

How can researchers assess the potential antimicrobial activity of Macaca nemestrina RNASE9?

Given that human RNASE9 demonstrates antibacterial activity against E. coli , a comprehensive assessment of Macaca nemestrina RNASE9's antimicrobial properties should include:

1. Radial Diffusion Assay:

• Prepare agarose plates containing target microorganisms

• Create wells in the agarose and add different concentrations of purified RNASE9

• Incubate for 18-24 hours and measure zones of inhibition

• Include appropriate positive controls (known antimicrobial peptides) and negative controls

2. Broth Microdilution Assay:

• Prepare serial dilutions of RNASE9 in microplate wells

• Add standardized bacterial suspensions (e.g., E. coli, Staphylococcus aureus)

• Incubate for 16-24 hours and measure optical density

• Calculate minimum inhibitory concentration (MIC) values

3. Time-Kill Kinetics:

• Incubate bacteria with RNASE9 at different concentrations

• Remove aliquots at various time points (0, 30, 60, 120, 180 minutes)

• Plate dilutions on appropriate agar media and count colonies

• This approach reveals whether RNASE9 exhibits concentration/time-dependent antibacterial activity as observed with human RNASE9

4. Membrane Permeabilization Assays:

• Use fluorescent dyes like propidium iodide or SYTOX Green

• Treat bacteria with RNASE9 and monitor dye uptake

• Measure fluorescence to quantify membrane disruption

5. Comparative Analysis:

• Test activity against a panel of microorganisms (Gram-positive bacteria, Gram-negative bacteria, fungi)

• Compare activity spectrum with human RNASE9

• Analyze the effect of ionic strength, pH, and temperature on antimicrobial activity

These methodologies provide comprehensive insights into Macaca nemestrina RNASE9's antimicrobial potential and its possible role in host defense within the male reproductive tract.

What structural analyses can be performed to determine the three-dimensional structure of Macaca nemestrina RNASE9?

To elucidate the three-dimensional structure of Macaca nemestrina RNASE9, researchers should employ complementary structural biology techniques:

1. X-ray Crystallography:

• Express and purify large quantities (>10 mg) of homogeneous RNASE9

• Screen various conditions to obtain protein crystals

• Collect diffraction data at synchrotron radiation facilities

• Process data and solve the structure by molecular replacement using human RNASE structures as templates

• Refine the structure to generate a high-resolution model

2. Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Prepare isotopically labeled RNASE9 (15N, 13C)

• Collect multi-dimensional NMR spectra

• Assign backbone and side-chain resonances

• Generate distance restraints and calculate the solution structure

• This approach is particularly useful for analyzing flexible regions

3. Computational Approaches:

• Homology Modeling:

  • Use human RNASE9 or other RNase A family members as templates

  • Generate multiple models and evaluate using energy minimization

  • Validate models using molecular dynamics simulations

• Ab initio Protein Structure Prediction:

  • Use methods like AlphaFold2 or RoseTTAFold

  • Generate predictions based solely on the amino acid sequence

4. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Expose RNASE9 to deuterium-containing buffers

• Analyze the rate of hydrogen-deuterium exchange by mass spectrometry

• Map regions of structural flexibility and solvent accessibility

5. Circular Dichroism (CD) Spectroscopy:

• Analyze secondary structure content (α-helices, β-sheets)

• Study thermal stability and folding properties

• Compare with human RNASE9 to identify structural differences

The combination of these methodologies would provide comprehensive insights into Macaca nemestrina RNASE9's three-dimensional structure, revealing important features that may correlate with its functional properties and evolutionary adaptations.

How do post-translational modifications influence the function of RNASE9 in primates?

Post-translational modifications (PTMs) can significantly influence protein structure, localization, stability, and function. For RNASE9, potential PTMs and their functional implications include:

1. N-Glycosylation:

• Many secreted proteins, including RNases, undergo N-glycosylation

• Potential N-glycosylation sites can be predicted from the amino acid sequence (Asn-X-Ser/Thr motifs)

• Glycosylation affects:

  • Protein folding and stability

  • Protection from proteolytic degradation

  • Recognition by cell surface receptors

2. Disulfide Bond Formation:

• RNase A family members typically contain multiple disulfide bonds

• These are critical for maintaining tertiary structure

• Correct disulfide bond formation is essential for proper folding and function

3. Proteolytic Processing:

• Signal peptide cleavage is essential for secretion

• Additional proteolytic processing may occur in the epididymal environment

• The N-terminal amino acid sequences of mature RNASE9 provide important information about processing events

Methodological Approaches to Study PTMs in RNASE9:

• Mass Spectrometry-Based Approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Glycoproteomics for detailed glycan structure analysis

  • Phosphoproteomics for identification of phosphorylation sites

• Site-Directed Mutagenesis:

  • Mutate potential modification sites

  • Compare functional properties of wild-type and mutant proteins

  • Assess the impact on antimicrobial activity and sperm binding

• Enzymatic Deglycosylation:

  • Treat native RNASE9 with enzymes like PNGase F or Endo H

  • Compare properties of glycosylated and deglycosylated forms

Understanding PTMs of RNASE9 in different primate species would provide valuable insights into the evolution of this protein and how structural modifications contribute to species-specific functions in reproductive biology and host defense.

What are the recommended approaches for generating antibodies against Macaca nemestrina RNASE9 for immunolocalization studies?

Based on successful approaches used for human RNASE9 , the following comprehensive protocol is recommended for generating antibodies against Macaca nemestrina RNASE9:

1. Antigen Preparation:

A. Recombinant Full-Length Protein:

• Express recombinant Macaca nemestrina RNASE9 in E. coli or mammalian cells

• Purify to >95% homogeneity using appropriate chromatography techniques

• Verify protein identity by mass spectrometry or N-terminal sequencing

B. Synthetic Peptides:

• Design 15-20 amino acid peptides from predicted antigenic regions

• Consider using multiple peptides targeting different regions

• Conjugate to carrier proteins (KLH or BSA) to enhance immunogenicity

2. Immunization Protocol:

This immunization schedule follows the protocol successfully used for human RNASE9 .

3. Antibody Characterization:

A. Specificity Testing:

• Western blot analysis against recombinant RNASE9

• Cross-reactivity assessment with other RNase family members

• Pre-absorption controls with immunizing antigen

B. Sensitivity Determination:

• Limit of detection in ELISA and Western blot

• Immunoprecipitation efficiency

C. Functional Validation:

• Immunofluorescence on known RNASE9-expressing tissues

• Blocking experiments to confirm specificity

4. Antibody Purification:

A. Polyclonal Antibodies:

• Protein A/G affinity purification

• Antigen-specific affinity purification for highest specificity

B. Monoclonal Antibodies (if developed):

• Protein A/G chromatography

• Ion exchange chromatography for additional purification

This approach should yield high-quality antibodies suitable for immunolocalization studies of Macaca nemestrina RNASE9, allowing researchers to map its distribution in reproductive tissues and on spermatozoa.

How can researchers investigate the evolutionary conservation of RNASE9 function across different primate species?

Investigating the evolutionary conservation of RNASE9 requires a multifaceted approach combining comparative genomics, biochemistry, structural biology, and functional assays:

1. Comparative Sequence Analysis:

A. Sequence Collection and Alignment:

• Retrieve RNASE9 sequences from various primate species

• Perform multiple sequence alignment using tools like MUSCLE or CLUSTAL

• Calculate sequence identity and similarity percentages

B. Evolutionary Rate Analysis:

• Calculate dN/dS ratios to identify sites under positive or purifying selection

• Identify lineage-specific accelerated evolution

• Map evolutionary changes onto the protein structure

2. Functional Comparison:

A. Recombinant Protein Production:

• Express and purify RNASE9 from multiple primate species

• Ensure comparable purity and structural integrity

B. Comparative Antimicrobial Assays:

• Test antimicrobial activity against the same panel of microorganisms

• Compare minimum inhibitory concentrations and killing kinetics

• Evaluate the spectrum of antimicrobial activity

C. Sperm Binding Assays:

• Develop cross-species sperm binding assays

• Assess whether RNASE9 from one species can bind to sperm from other species

• Quantify binding affinities and specificities

3. Expression Pattern Comparison:

A. Tissue Distribution Analysis:

• Compare RNASE9 expression patterns across primate species using:

  • RT-PCR or RNA-Seq for transcript detection

  • Immunohistochemistry for protein localization

• Focus on reproductive tract tissues and potential extragonadal sites

4. Interspecies Complementation Experiments:

• Express RNASE9 from different species in the same cellular background

• Compare subcellular localization and secretion efficiency

• Assess functional readouts (e.g., antimicrobial protection)

This comprehensive approach would provide significant insights into the evolutionary conservation and divergence of RNASE9 function across primate species, highlighting both conserved ancestral functions and species-specific adaptations.

How does Macaca nemestrina RNASE9 compare to other members of the RNase A superfamily in terms of structure and function?

The RNase A superfamily consists of diverse members with varying functions. Comparing Macaca nemestrina RNASE9 to other family members reveals important structural and functional relationships:

Structural Comparisons:

• Catalytic Residues:

  • Traditional RNases (RNase 1-5) contain conserved catalytic residues (His12, Lys41, His119 in RNase A numbering)

  • RNASE9 lacks these conserved catalytic residues, explaining its absence of ribonucleolytic activity

  • This loss of catalytic function is similar to RNases 9-13, which have evolved alternative functions

• Disulfide Bonding Pattern:

  • RNase A family members typically contain conserved disulfide bonds

  • Analysis of disulfide bonding patterns can reveal structural constraints maintained during evolution

Functional Comparisons:

This comparative analysis highlights how RNASE9 represents an example of functional diversification within the RNase A superfamily, evolving specialized roles in reproduction and host defense despite losing ancestral catalytic function.

What experimental designs can be used to study the interaction between Macaca nemestrina RNASE9 and spermatozoa?

To investigate the interaction between Macaca nemestrina RNASE9 and spermatozoa, researchers should consider the following experimental approaches:

1. Binding Studies:

A. Direct Binding Assays:

• Label recombinant RNASE9 with fluorescent tags or biotin

• Incubate with spermatozoa under various conditions

• Analyze binding using flow cytometry or fluorescence microscopy

• Quantify binding parameters (affinity, saturation)

B. Competition Assays:

• Use unlabeled RNASE9 to compete with labeled RNASE9

• Identify specific binding sites through selective inhibition

• Test fragments or mutants of RNASE9 to map binding domains

2. Immunolocalization:

A. Confocal Microscopy:

• Incubate fixed sperm with anti-RNASE9 antibodies

• Use fluorescently-labeled secondary antibodies for detection

• Perform z-stack imaging to precisely locate RNASE9 on sperm surfaces

• Co-stain with markers for different sperm compartments (acrosome, mitochondria)

B. Immunoelectron Microscopy:

• Localize RNASE9 at ultrastructural level using gold-labeled antibodies

• Identify potential membrane microdomains associated with RNASE9

3. Functional Studies:

A. Sperm Function Assays:

• Assess the effect of RNASE9 on:

  • Sperm motility parameters using computer-assisted sperm analysis

  • Capacitation using chlortetracycline staining or tyrosine phosphorylation

  • Acrosome reaction using fluorescent lectins

B. Antibody Blocking Studies:

• Use anti-RNASE9 antibodies to block endogenous RNASE9

• Assess the impact on sperm function parameters

4. Receptor Identification:

A. Cross-linking Studies:

• Use chemical cross-linkers to capture RNASE9-receptor complexes

• Identify binding partners by mass spectrometry

B. Yeast Two-Hybrid or Mammalian Two-Hybrid Screens:

• Identify proteins that interact with RNASE9

• Confirm interactions using co-immunoprecipitation

5. Real-time Interaction Analysis:

A. Surface Plasmon Resonance (SPR):

• Immobilize RNASE9 or potential binding partners

• Measure binding kinetics and affinity constants

• Determine the effect of pH, ionic strength, and temperature on binding

These methodological approaches would provide comprehensive insights into the interaction between Macaca nemestrina RNASE9 and spermatozoa, elucidating both the binding mechanisms and functional consequences of this interaction.

What strategies can be employed to study the potential role of Macaca nemestrina RNASE9 in host defense against reproductive tract infections?

To investigate the role of Macaca nemestrina RNASE9 in host defense against reproductive tract infections, researchers should consider the following comprehensive approaches:

1. In Vitro Antimicrobial Assays:

A. Activity Against Reproductive Tract Pathogens:

• Test antimicrobial activity against common reproductive tract pathogens:

  • Bacteria (Escherichia coli, Staphylococcus aureus, Neisseria gonorrhoeae)

  • Fungi (Candida albicans)

  • Viruses (Herpes simplex virus)

• Determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs)

B. Mechanism of Action Studies:

• Membrane permeabilization assays using fluorescent dyes

• Electron microscopy to visualize microbial cell damage

• Transcriptomics to identify affected pathways in microorganisms

2. Ex Vivo Studies:

A. Reproductive Tract Explant Models:

• Culture epididymal tissue explants from Macaca nemestrina

• Challenge with pathogens in the presence or absence of RNASE9

• Assess microbial growth and tissue inflammation markers

B. Sperm Protection Assays:

• Incubate spermatozoa with pathogens with or without RNASE9

• Evaluate sperm viability, motility, and functional parameters

• Determine if RNASE9 protects sperm from pathogen-induced damage

3. Expression Studies During Infection:

A. Infection Models:

• Analyze RNASE9 expression in response to:

  • Bacterial lipopolysaccharide (LPS) stimulation

  • Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6)

  • Viral mimetics (poly I:C)

• Use both cell culture and tissue explant models

B. Clinical Samples (if available):

• Compare RNASE9 levels in healthy vs. infected reproductive tract samples

• Correlate RNASE9 expression with pathogen load or inflammatory markers

4. Cell Culture Models:

A. Epithelial Cell Defense:

• Transfect reproductive tract epithelial cells to express RNASE9

• Challenge with pathogens and assess survival/infection rates

• Measure inflammatory cytokine production

B. Immune Cell Interaction:

• Study the effect of RNASE9 on neutrophil and macrophage function

• Assess chemotactic properties and enhancement of phagocytosis

• Evaluate modulation of immune cell cytokine production

5. In Vivo Models (where ethically appropriate):

A. Expression Analysis:

• Induce experimental infections in the reproductive tract

• Monitor RNASE9 expression using qPCR and immunohistochemistry

• Correlate with immune cell infiltration and pathogen clearance

These methodological approaches would provide comprehensive insights into the potential role of Macaca nemestrina RNASE9 in host defense against reproductive tract infections, particularly its direct antimicrobial activities and potential immunomodulatory functions.