Recombinant Human Inactive serine protease 54 (PRSS54)

What is PRSS54 and what are its key structural features?

PRSS54 is a putative serine-type endopeptidase containing the peptidase S1 domain. The human gene encodes a protein that undergoes alternative splicing, resulting in multiple transcript variants . It belongs to the larger family of serine proteases, which constitute nearly one-third of all proteases. The protein is also known by several aliases including Cancer/testis antigen 67 (CT67), Inactive serine protease 54, and Plasma kallikrein-like protein 4 . PRSS54 exhibits unique structural characteristics, including a change in protein size from approximately 50 kDa in testis tissue to 42 kDa in mature sperm, suggesting post-translational modifications during sperm maturation .

How is PRSS54 conserved across species and what does this suggest about its function?

Mouse and human PRSS54 proteins share significant sequence homology, with approximately 68% antigen sequence identity between human PRSS54 and its mouse and rat orthologs . This evolutionary conservation suggests fundamental biological importance. The conserved nature of PRSS54 across mammalian species indicates its likely essential role in reproductive processes. Functional studies in mouse models have confirmed that despite some species differences, the core functions in acrosome biogenesis and sperm function appear to be maintained, making mouse models valuable for understanding human PRSS54 functions .

What are the optimal expression systems for producing functional recombinant PRSS54 for research purposes?

For experimental studies requiring recombinant PRSS54, researchers have successfully used both bacterial and mammalian expression systems, though with different advantages. While bacterial systems (such as E. coli BL21 strains) can produce higher protein yields, mammalian cell lines like HEK293 provide more accurate post-translational modifications . For structural and functional studies of PRSS54, the recommended approach is expression in mammalian systems to preserve native glycosylation patterns and ensure proper protein folding. Key methodological steps include:

• Cloning the PRSS54 coding sequence into an appropriate expression vector with a purification tag (His, GST, or Fc)

• Transfection into HEK293 cells for mammalian expression

• Purification using affinity chromatography based on the chosen tag

• Validation of protein purity by SDS-PAGE (≥85% purity recommended)

What experimental approaches are most effective for studying PRSS54's role in acrosome formation?

To investigate PRSS54's function in acrosome formation, researchers should employ a multi-faceted approach combining genetic manipulation with advanced microscopy techniques. Based on recent research methodology , the following experimental design has proven effective:

• Generate PRSS54 knockout models in mice using CRISPR/Cas9 technology

• Employ transmission electron microscopy (TEM) to analyze ultrastructural changes in developing spermatids

• Use immunofluorescence microscopy with co-localization studies to track PRSS54 and interacting acrosomal proteins during sperm development

• Perform sperm function tests including zona pellucida penetration assays to correlate structural changes with functional outcomes

• Conduct rescue experiments using transgenic expression of wild-type PRSS54 to confirm phenotype specificity

This comprehensive approach has successfully revealed PRSS54's temporal localization pattern and its critical role in acrosomal granule positioning and membrane fusion events .

What proteins interact with PRSS54 during acrosome biogenesis and what experimental approaches best characterize these interactions?

PRSS54 has been shown to interact with several key acrosomal proteins, including ZPBP1, ZPBP2, ACRBP, and ZP3R . These interactions appear crucial for proper acrosome formation and sperm-egg interaction. To effectively characterize these protein-protein interactions, researchers should employ:

• Co-immunoprecipitation assays with antibodies against PRSS54 followed by mass spectrometry

• Proximity ligation assays to confirm interactions in situ

• Yeast two-hybrid or mammalian two-hybrid systems for direct interaction screening

• FRET (Förster Resonance Energy Transfer) analysis for dynamic interaction studies

• Comparative distribution analysis of these proteins in wild-type versus PRSS54-deficient models

How can researchers effectively measure PRSS54 enzymatic activity and what are the key considerations?

Despite being classified as an "inactive" serine protease, functional analysis of PRSS54's potential enzymatic activity requires careful experimental design. Based on methodologies used for related serine proteases , researchers should consider:

• Using synthetic fluorescent substrates such as N-t-Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC)

• Conducting assays at physiologically relevant conditions (37°C, pH 8.0)

• Including appropriate controls with known serine protease inhibitors (PMSF, aprotinin, α1-antitrypsin)

• Performing kinetic studies to determine potential catalytic efficiency (kcat/Km)

• Comparing wild-type PRSS54 with site-directed mutants of the catalytic triad

While current evidence suggests limited proteolytic activity, these approaches would help clarify whether PRSS54 possesses context-specific enzymatic functions or primarily acts through protein-protein interactions.

What techniques are most effective for analyzing PRSS54 expression patterns during spermatogenesis?

To accurately characterize PRSS54 expression during different stages of spermatogenesis, researchers should employ a combination of:

• Single-cell RNA sequencing to identify cell-specific expression patterns

• In situ hybridization to localize mRNA expression in testicular tissues

• Stage-specific isolation of spermatogenic cells followed by qRT-PCR

• Immunohistochemistry with validated antibodies to detect protein expression

• Western blotting on fractionated testicular samples to track protein size changes (from 50kDa to 42kDa) during sperm maturation

This multi-technique approach helps resolve temporal and spatial expression patterns crucial for understanding PRSS54's developmental regulation.

How do alternative splicing events affect PRSS54 function and how can these variants be systematically studied?

PRSS54 undergoes alternative splicing resulting in multiple transcript variants . To systematically investigate these variants and their functional implications, researchers should:

• Employ RNA-Seq analysis of testicular tissue to identify all splice variants

• Design variant-specific PCR primers for quantitative expression analysis

• Generate expression constructs for each major variant

• Perform functional rescue experiments with individual variants in PRSS54-deficient models

• Use domain-specific antibodies to detect protein isoforms in different cellular compartments

This approach would help determine whether specific splice variants have distinct functions during different stages of spermatogenesis or in different subcellular locations.

What are the complete phenotypic manifestations of PRSS54 deficiency in model organisms?

PRSS54 deficiency in mouse models produces multiple phenotypic manifestations affecting reproductive and potentially other physiological systems. The comprehensive phenotypic profile includes:

This comprehensive phenotypic analysis suggests PRSS54 may have broader physiological roles beyond reproduction, warranting further investigation in other organ systems .

What is the potential relationship between PRSS54 polymorphisms and male infertility in humans?

While direct evidence linking PRSS54 polymorphisms to human male infertility is still emerging, the research findings in mouse models strongly suggest potential clinical relevance. To investigate this relationship, researchers should:

• Perform targeted sequencing of PRSS54 in infertile male cohorts versus fertile controls

• Apply polygenic risk score (PRS) methodologies to identify potential contributions to infertility phenotypes

• Conduct functional studies on identified variants using cellular models

• Analyze sperm from men with PRSS54 variants for acrosomal abnormalities

• Consider PRSS54 in multi-gene panels for clinical infertility screening

The integration of these approaches with advanced statistical methods like those described for polygenic risk scores could improve prediction accuracy for male infertility and potentially guide personalized treatment approaches .

How does PRSS54 function compare with other testis-specific serine proteases and what are the implications for reproductive biology?

PRSS54 belongs to a larger family of serine proteases, many of which have specific roles in reproduction. Unlike highly active proteases such as acrosin, PRSS54 appears to function more through protein-protein interactions despite its protease domain. Key comparative insights include:

• PRSS54 shows structural similarity to the matriptase subfamily of transmembrane serine proteases (TTSPs)

• Unlike polyserase-I, which can generate independent serine protease domains that function independently, PRSS54 appears to function as a single unit

• While many serine proteases participate actively in zona pellucida penetration through proteolytic activity, PRSS54 appears to function primarily in acrosome biogenesis and structural organization

• PRSS54's "inactive" classification may reflect context-specific activity rather than complete lack of enzymatic function

This comparative analysis highlights the diverse mechanisms by which serine proteases contribute to reproductive processes, with PRSS54 potentially representing a structural or regulatory member of this family rather than a primarily catalytic one.

What methodological approaches best distinguish between enzymatic and non-enzymatic functions of serine proteases like PRSS54?

Distinguishing between enzymatic and non-enzymatic functions of serine proteases requires sophisticated experimental approaches:

• Generate catalytic triad mutants that lack enzymatic activity but maintain structural integrity

• Perform complementation studies using chimeric proteins with the catalytic domain from related active proteases

• Use activity-based protein profiling with serine protease-specific probes

• Employ proximity labeling methods (BioID, APEX) to identify interaction partners independent of enzymatic activity

• Conduct structural studies (X-ray crystallography, cryo-EM) to identify potential substrate binding sites and conformational changes

These approaches would help determine whether PRSS54's role in acrosome formation depends on proteolytic activity or primarily structural interactions with binding partners like ZPBP1, ZPBP2, ACRBP, and ZP3R .

What emerging technologies could advance our understanding of PRSS54's role in reproductive biology?

Several cutting-edge technologies offer promising avenues for deeper insights into PRSS54 function:

• Spatial transcriptomics to map expression patterns with unprecedented resolution

• CRISPR-based epigenome editing to study regulation of PRSS54 expression

• High-resolution structural biology techniques (cryo-EM, AlphaFold) to elucidate protein structure

• Organoid models of testicular development to study PRSS54 in a physiologically relevant context

• Single-molecule imaging to track PRSS54 dynamics during acrosome biogenesis

• Multi-omics integration approaches to place PRSS54 in broader signaling networks

These technologies would enable researchers to address current knowledge gaps regarding PRSS54's precise molecular mechanisms and regulatory pathways.

How might PRSS54 research contribute to advances in male contraception or fertility treatments?

Given PRSS54's specific expression in testis and critical role in sperm function, it represents a potential target for reproductive interventions:

• Development of small molecule inhibitors specific to PRSS54 could provide a non-hormonal male contraceptive approach

• Diagnostic assays measuring PRSS54 levels or detecting antibodies against PRSS54 could help identify certain causes of male infertility

• Gene therapy approaches to restore functional PRSS54 expression might address specific forms of infertility

• Recombinant PRSS54 could potentially improve sperm function in assisted reproductive technologies

• PRSS54-based immunocontraceptive vaccines represent another potential application

Researchers pursuing these directions should carefully consider the broader physiological roles of PRSS54, as mouse models suggest potential functions beyond reproduction that could lead to unintended side effects of therapeutic interventions .