Recombinant Rat Inactive serine protease 35 (Prss35)
Description
Introduction
Recombinant Rat Inactive serine protease 35 (Prss35) is a serine protease that has garnered interest for its involvement in various biological processes, including female reproduction, tumor progression, and response to hyperosmotic stress . Prss35 is expressed in the ovary and early pregnant uterus, suggesting a role in oocyte development, ovulation, implantation, and decidualization, but studies in mice have not confirmed this hypothesis . Recent research has identified Prss35 as a key regulator of the matrisome under hyperosmotic stress conditions and has shown its downregulation in hepatocellular carcinoma (HCC), suggesting its potential as a prognostic biomarker .
Gene and Protein Characteristics
PRSS35 encodes for serine protease 35, and the human gene is located on chromosome 6q and has been linked to cleft lip/palate, although studies in Prss35 knockout mice did not show these defects . The protein has multiple predicted cleavage sites, and studies show that mutations at these sites disrupt Prss35 cleavage, which suggests that cleavage at these sites is required for subsequent proteolytic processing .
Expression and Localization
Prss35 exhibits distinct spatiotemporal expression patterns in various tissues.
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Uterus: Undetectable in the wild-type (WT) uterus on gestation day 3.5, Prss35 is detected in the stromal compartment surrounding the embryo on day 4.5 . Its expression shifts towards the mesometrial side of the stromal compartment from days 5.5 to 7.5. Expression remains detectable at lower levels in the stromal compartment on the mesometrial side of the day 7.5 WT uterus but is undetectable in the embryo from days 4.5 to 7.5 .
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Ovary: Prss35 is expressed in granulosa cells and the corpus luteum of the ovary . It is also expressed in forming and regressing corpora lutea (CL) .
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Tumors: Prss35 is selectively upregulated in high-grade human squamous cell carcinomas (SCCs) .
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Liver: PRSS35 protein levels were markedly reduced in PLC, HepG2, and Hep3B liver cancer cells, relative to its accumulation in THLE3 cells .
Functional Studies
Research has explored the functional roles of Prss35 in different contexts.
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Reproduction: Studies in Prss35 knockout mice showed that superovulation of immature females produced comparable numbers of cumulus-oocyte complexes compared to wild-type mice. The number of implantation sites detected on days 4.5 and 7.5 were comparable, and there were no obvious differences in the expression of implantation and decidualization marker genes in uteri on days 4.5 or 7.5 . Comparable litter sizes from WT × WT and (−/−) Prss35 (−/−)× Prss35 were also observed .
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Tumorigenesis: Ablation of PRSS35 in mouse models of wound- or chemically-induced tumorigenesis resulted in aberrant collagen composition in the ECM . PRSS35 KO skin showed a more dense collagen matrix, with more collagen per area in scars and papillomas and fewer thin collagen fibers compared to thick collagen fibers .
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Response to Hyperosmotic Stress: PRSS35 regulates the matrisome under hyperosmotic stress conditions. Exposure of skin fibroblasts to hyperosmotic stress regulates the expression of genes, which help cells to cope with the stress condition . PRSS35 interacts with collagens and collagen-associated proteins in the secretory pathway of fibroblasts and affects the extracellular matrix proteome, which limits cell proliferation .
Role in Hepatocellular Carcinoma (HCC)
PRSS35 was identified as the most significantly downregulated protein in the PLC secretome . Both intracellular and extracellular PRSS35 protein levels were markedly reduced in PLC, HepG2, and Hep3B liver cancer cells, relative to its accumulation in THLE3 cells . Lower levels of truncated PRSS35 forms were observed in HCC patient serum compared to normal subjects, suggesting that secreted PRSS35 protein could serve as a potential prognostic biomarker for HCC patients .
HNF4A is a potential transcriptional factor regulating PRSS35 . Both PRSS35 protein and mRNA levels were downregulated when HNF4A was knocked down in HepG2 cells and upregulated with HNF4A overexpression .
Tables
Table 1: Expression of Prss35 in Uterine Tissue
| Tissue | Gestation Day | Expression |
|---|---|---|
| Wild-type (WT) uterus | 3.5 | Undetectable |
| WT stromal compartment surrounding embryo | 4.5 | Detected |
| WT stromal compartment | 5.5-7.5 | Strong staining shifted to the stromal compartment on the mesometrial side. The expression level remained detectable at a lower level in the stromal compartment on the mesometrial side of the day 7.5 WT uterus. |
| Embryo | 4.5-7.5 | Undetectable |
Product Specs
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Target Background
KEGG: rno:315866
UniGene: Rn.161916
Q&A
What is rat PRSS35 and how does it compare to other serine proteases?
PRSS35 belongs to the trypsin-like serine protease family but is considered a pseudo serine protease. Unlike conventional serine proteases that contain the catalytic triad of histidine, aspartic acid, and serine residues, PRSS35 has structural modifications that affect its catalytic activity. While many serine proteases are membrane-anchored through various mechanisms (transmembrane domains or GPI linkages), PRSS35 is secreted and functions in the extracellular environment . Research comparing rat and human PRSS35 has shown conservation of key structural elements, though species-specific differences in glycosylation patterns and processing may exist.
What are the key structural characteristics of recombinant rat PRSS35?
Recombinant rat PRSS35 exists in multiple forms, with full-length PRSS35 (FL-PRSS35) having a distinctly higher molecular weight than the secreted form (SF-PRSS35) . The protein undergoes processing via cleavage by proprotein convertases, which is critical for its biological activity. Western blot analysis using antibodies against the N-terminus of PRSS35 can differentiate between these forms, with the full-length protein being predominant in cell lysates while the shorter form is found in the secreted fraction . The mature secreted form contains the active domain required for its proteolytic functions.
How should researchers verify the identity and purity of recombinant rat PRSS35?
Verification of recombinant rat PRSS35 requires multiple analytical approaches:
| Verification Method | Purpose | Expected Results |
|---|---|---|
| SDS-PAGE | Molecular weight confirmation | ~42-45 kDa (FL-PRSS35); ~30-35 kDa (SF-PRSS35) |
| Western Blot | Specific protein identification | Bands corresponding to FL-PRSS35 and SF-PRSS35 |
| Mass Spectrometry | Peptide sequence confirmation | Matches to rat PRSS35 sequence with potential post-translational modifications |
| Activity Assay | Functional verification | Cleavage of synthetic substrates containing KK motifs |
Additionally, researchers should confirm proper folding using circular dichroism spectroscopy and assess glycosylation status using glycosidase treatments followed by gel mobility shift analysis.
What expression systems are optimal for producing recombinant rat PRSS35?
For functional studies of rat PRSS35, the choice of expression system is critical. Mammalian expression systems like HEK293 or CHO cells are preferred over bacterial systems due to their ability to perform post-translational modifications, particularly glycosylation, which may be essential for proper folding and activity . When designing expression constructs, researchers should consider including:
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A strong promoter (CMV for mammalian cells)
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Signal peptide for secretion
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Fusion tags for purification (His-tag or Fc-tag)
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Protease cleavage sites for tag removal
Transient transfection typically yields sufficient protein for preliminary studies, while stable cell lines are recommended for larger-scale production and consistency across experiments.
What challenges arise during purification of recombinant rat PRSS35?
Purification of recombinant rat PRSS35 presents several challenges that researchers should anticipate:
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Differentiating between processed (active) and unprocessed forms
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Preserving proteolytic activity during purification
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Removing contaminant proteases that might cleave PRSS35
A recommended purification protocol would involve:
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Collection of conditioned medium from expressing cells
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Affinity chromatography using tag-specific resins
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Ion exchange chromatography for further purification
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Size exclusion chromatography to separate different forms
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Activity-based verification using synthetic substrates
Researchers should particularly note that the full-length and processed forms have distinct molecular weights as observed in Western blot analyses of PRSS35 overexpression systems .
How can researchers effectively measure the proteolytic activity of PRSS35?
Despite being classified as a pseudo serine protease, PRSS35 demonstrates specific proteolytic activity against targets like CXCL2 through recognition of a tandem lysine (KK) motif . To measure this activity:
| Assay Type | Methodology | Applications |
|---|---|---|
| Fluorogenic Substrate Assay | Synthetic peptides with KK motifs and fluorogenic leaving groups | Quantitative activity measurement |
| In vitro Cleavage Assay | Incubation with recombinant substrates followed by SDS-PAGE analysis | Substrate specificity analysis |
| Cell-based Assay | Co-expression of PRSS35 with potential substrates | Cellular context validation |
| Mass Spectrometry | Identification of cleavage sites in digested substrates | Precise mapping of substrate recognition |
Control experiments should include heat-inactivated PRSS35 and protease inhibitor treatments to confirm specificity of observed proteolytic events.
What methodologies are recommended for studying PRSS35's role in tumor suppression?
To investigate the tumor suppression function of PRSS35, researchers can employ several complementary approaches:
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In vitro cell proliferation and migration assays comparing PRSS35-expressing and control cells
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Xenograft models with differential PRSS35 expression to assess tumor growth kinetics
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Analysis of neutrophil recruitment to tumor sites using immunohistochemistry
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Quantification of neutrophil extracellular traps (NETs) in response to PRSS35 expression
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CXCL2 level measurements by ELISA before and after PRSS35 treatment
Research has demonstrated that PRSS35 suppresses hepatocellular carcinoma progression by cleaving CXCL2, which attenuates neutrophil recruitment to tumors and formation of neutrophil extracellular traps . Similar mechanisms could be explored in other cancer models using rat PRSS35.
How does PRSS35 interact with CXCL2 and what techniques can detect this interaction?
PRSS35 targets CXCL2 through a specific recognition of the tandem lysine (KK) motif . Researchers investigating this interaction should consider:
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Co-immunoprecipitation assays to confirm direct binding
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Surface plasmon resonance (SPR) to determine binding kinetics
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FRET-based approaches for real-time interaction studies
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In vitro cleavage assays using site-directed mutagenesis of the KK motif
The interaction leads to CXCL2 degradation, which can be monitored by Western blot analysis or mass spectrometry to identify specific cleavage products. When designing experiments, researchers should control for potential confounding factors like non-specific proteolysis and ensure physiologically relevant concentrations of both proteins.
What downstream signaling pathways are affected by PRSS35 activity?
PRSS35-mediated degradation of CXCL2 affects neutrophil recruitment and NET formation, which impacts tumor progression . To study these downstream effects, consider:
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Phosphorylation status of CXCR2 (CXCL2 receptor) using phospho-specific antibodies
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Activation of downstream MAP kinase cascades
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NF-κB signaling pathway activity
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Cytokine profiling of tumor microenvironment
These analyses help elucidate how PRSS35 proteolytic activity translates into altered cellular responses and tissue-level changes in disease models.
What controls are essential when working with recombinant rat PRSS35?
Rigorous experimental controls are critical when working with PRSS35:
| Control Type | Purpose | Implementation |
|---|---|---|
| Catalytic-dead mutant | Control for non-proteolytic functions | Site-directed mutagenesis of catalytic residues |
| Heat-inactivated protein | Control for contaminant activities | Heating at 95°C for 10 minutes |
| Protease inhibitor panel | Specificity confirmation | Include serine protease inhibitors (PMSF, aprotinin) |
| Species-matched control protein | Control for general protein effects | Another recombinant protein of similar size |
| Vehicle control | Control for buffer effects | Matching buffer composition without protein |
Additionally, concentration-response relationships should be established for all observed effects to confirm biological relevance.
How should researchers address contradictory findings about PRSS35 function?
When encountering contradictory results regarding PRSS35 function, implement this systematic approach:
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Verify protein identity and activity using multiple methods
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Compare experimental conditions across studies (cell types, cancer models, etc.)
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Consider context-dependent effects based on microenvironment
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Assess potential differences between human and rat PRSS35
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Examine splice variants or post-translational modifications
In hepatocellular carcinoma, PRSS35 has been identified as a tumor suppressor , but its role may differ in other tissues or disease contexts. Careful documentation of experimental conditions and cellular contexts will help resolve apparent contradictions.
How can CRISPR-Cas9 technology be applied to study PRSS35 function?
CRISPR-Cas9 offers powerful approaches for PRSS35 research:
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Generate PRSS35 knockout cell lines or animal models
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Create knock-in models with tagged or mutant PRSS35
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Perform domain-specific mutations to map functional regions
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Implement CRISPRi/CRISPRa for controlled expression modulation
When designing guide RNAs, consider targeting conserved regions to ensure complete loss of function, and implement validation strategies including sequencing and protein expression analysis. For studying processing mechanisms, CRISPR editing of cleavage sites can provide insights into the importance of proprotein convertase processing for PRSS35 activity.
What are the emerging technologies for studying PRSS35 in complex tissue environments?
Recent technological advances offer new possibilities for PRSS35 research:
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Spatial transcriptomics to map PRSS35 expression in tissue contexts
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Single-cell proteomics to identify cell populations expressing PRSS35
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Intravital microscopy to visualize PRSS35-mediated neutrophil recruitment in real-time
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Activity-based protein profiling to detect active PRSS35 in complex samples
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Organoid models to study PRSS35 function in 3D tissue architecture
These approaches address limitations of traditional in vitro systems by preserving the complexity of the tumor microenvironment where PRSS35 mediates its effects on neutrophil recruitment and NET formation .
What are the critical knowledge gaps in PRSS35 research?
Despite progress in understanding PRSS35, several knowledge gaps remain:
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Complete substrate repertoire beyond CXCL2
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Regulatory mechanisms controlling PRSS35 expression
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Species-specific differences between human and rat PRSS35
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Potential roles in non-cancer pathologies
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Structural basis for substrate recognition and specificity
Addressing these gaps requires integrative approaches combining structural biology, proteomics, and in vivo functional studies. The tandem lysine (KK) recognition motif identified for CXCL2 cleavage provides a starting point for substrate prediction, but comprehensive substrate profiling is needed.
How might PRSS35 research translate to therapeutic applications?
The tumor suppressor function of PRSS35 in hepatocellular carcinoma suggests several translational research directions:
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Development of recombinant PRSS35 as a biotherapeutic
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Small molecule enhancers of endogenous PRSS35 expression
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Peptidomimetics that mimic PRSS35 substrate specificity
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Combined approaches targeting both PRSS35 and neutrophil function
Researchers pursuing translational applications should consider delivery challenges, potential immunogenicity, and off-target effects. Preliminary testing in patient-derived xenograft models would provide valuable insights into therapeutic potential before clinical development.
