SLURP1 Human Dimer

What is the molecular structure of SLURP1 Human Dimer?

SLURP1 Human Recombinant is produced as a non-glycosylated homodimer consisting of two chains spanning from Leu23 to Leu103, with each chain containing 89 amino acids including an 8 amino acid His tag at the N-terminus. The total calculated molecular mass for each chain is approximately 9.9kDa . SLURP1 belongs to the Ly6/uPAR family but notably lacks a GPI-anchoring signal sequence, which distinguishes it from other family members . The protein's amino acid sequence begins with MKHHHHHHHLK CYTCKEPMTS ASCRTITRCK PEDTACMTTL VTVEAEYPFN QSPV, including the His tag region . This dimeric structure is important to consider when designing experiments involving this protein.

How is SLURP1 expressed in different tissues and what are its primary biological functions?

SLURP1 serves multiple biological functions in different tissues. In the ocular surface, it functions as an immunomodulatory protein and is abundantly expressed in the corneal epithelium . Notably, Slurp1 transcripts are the 11th most abundant in the mouse cornea, indicating its significance in this tissue . In the skin, SLURP1 acts as a marker of late differentiation and is crucial for maintaining the physiological and structural integrity of keratinocyte layers .

Research has demonstrated that SLURP1 functions as:

• An anti-inflammatory molecule

• An anti-angiogenic factor

• A pro-differentiation factor that regulates cell cycle progression

• A modulator of cell adhesion and migration

These diverse functions highlight SLURP1's importance in maintaining tissue homeostasis in multiple organ systems.

How does SLURP1 expression vary during development and across different physiological conditions?

Developmental studies in mice have shown that Slurp1 expression follows a specific temporal pattern. It is undetectable in early embryonic stages (E13, E16) and in newborn mice (PN1), with barely detectable levels at postnatal day 10 (PN10) . Expression increases rapidly after eyelid opening and reaches stable levels around postnatal day 20 (PN20) . This pattern suggests that Slurp1 expression is developmentally regulated and coincides with corneal maturation.

Interestingly, SLURP1 expression does not vary significantly between different mouse strains (Balb/C, FVBN, C57Bl/6, and DBA/2J) or between genders . In humans, tear SLURP1 levels also do not show significant variation between genders or across different age groups (18-80 years) .

What are the molecular mechanisms by which SLURP1 regulates cell cycle progression?

SLURP1 functions as a pro-differentiation factor that specifically stalls the G1-S transition during cell cycle progression . Research using human corneal limbal epithelial (HCLE) cells engineered to express SLURP1 (HCLE-SLURP1) has revealed several key mechanisms:

• SLURP1 overexpression results in decreased expression of cyclins, particularly CCNE (Cyclin E) and CCND1/D2 (Cyclin D1/D2) .

• It downregulates cyclin-dependent kinases, including CDK4 and CDK6, which are critical for G1-S phase transition .

• SLURP1 upregulates the CDK inhibitor p15/CDKN2B, further contributing to cell cycle arrest .

These molecular changes culminate in:

• Decreased numbers of Ki67-positive cells (a marker of proliferation)

• Increased cell number doubling time

• Stalling in G1-S phase transition during the cell cycle

This mechanism explains the anti-proliferative effect of SLURP1 and suggests its potential utility in research focused on cell cycle regulation and differentiation processes.

What phenotypes are observed in SLURP1-null models, and what do they reveal about SLURP1 function?

Studies using Slurp1-null (Slurp1X−/−) mice have provided valuable insights into SLURP1 function in vivo. Comparison of 10-week-old wild type (WT) and Slurp1X−/− mouse corneas revealed several important phenotypic differences:

• Histological examination showed largely comparable corneal structure, except for a few loosely attached superficial cells in Slurp1X−/− corneas .

• Slurp1X−/− corneas exhibited increased numbers of Ki67-positive cells, indicating enhanced proliferation in the absence of SLURP1 .

• Expression and/or localization of tight junction proteins (Tjp1 and Pard3) and desmosomal protein (Dsp) were altered in Slurp1X−/− corneas .

• The mutant corneas displayed increased superficial fragility, suggesting compromised epithelial integrity .

• Slurp1X−/− corneas demonstrated slower corneal epithelial wound healing following experimental injury .

Interestingly, despite SLURP1's known anti-inflammatory and anti-angiogenic properties, naïve 10-week-old Slurp1X−/− corneas did not display spontaneous angiogenic inflammation . This observation suggests that corneal immune- and angiogenic-privilege is regulated by multiple redundant mechanisms, not solely dependent on SLURP1.

These phenotypes collectively indicate that SLURP1 plays crucial roles in corneal epithelial homeostasis, particularly in regulating proliferation, cell adhesion, and migration.

How does SLURP1 contribute to wound healing and epithelial barrier function?

SLURP1 plays a significant role in wound healing and maintenance of epithelial barrier function. Research comparing wild-type and Slurp1X−/− mouse corneas has demonstrated that:

• Slurp1X−/− corneas exhibit slower corneal epithelial wound healing following experimental epithelial debridement .

• The absence of SLURP1 leads to altered expression and/or localization of tight junction proteins (Tjp1 and Pard3) and desmosomal protein (Dsp), which are crucial for cell-cell adhesion and barrier function .

• Slurp1X−/− corneas display increased superficial fragility, suggesting compromised epithelial integrity .

These findings indicate that SLURP1 contributes to wound healing by regulating cell proliferation, differentiation, and the expression of adhesion molecules. The altered expression of junction proteins in Slurp1X−/− corneas suggests that SLURP1 is important for establishing and maintaining proper epithelial barrier function.

Researchers investigating wound healing mechanisms should consider SLURP1's role as a modulator of cell cycle progression and its influence on the expression of adhesion molecules, as these functions likely contribute to its effects on tissue repair and barrier integrity.

What are the recommended protocols for handling and storing recombinant SLURP1?

For optimal handling and storage of recombinant SLURP1, the following protocols are recommended based on commercial preparations:

• Storage of lyophilized protein: Store lyophilized SLURP1 at -20°C to maintain stability .

• Reconstitution protocol:

  • Add deionized water to prepare a working stock solution of approximately 0.5mg/ml

  • Allow the lyophilized pellet to dissolve completely

• Post-reconstitution handling:

  • Aliquot the reconstituted product to avoid repeated freezing/thawing cycles

  • Reconstituted protein can be stored at 4°C for a limited period (up to two weeks without showing changes)

• Sterility considerations: SLURP1 recombinant protein is typically not sterile. For cell culture applications, filter the product using an appropriate sterile filter before use .

• Formulation information: Commercial SLURP1 is typically filtered (0.4μm) and lyophilized from a 0.5mg/ml solution in 20mM Tris buffer and 50mM NaCl, pH 8.0 .

Following these handling and storage recommendations will help ensure the stability and activity of SLURP1 in experimental settings.

What methods are recommended for measuring SLURP1 levels in biological samples?

Several methods have been successfully employed to measure SLURP1 levels in biological samples:

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • This technique has been used to quantify SLURP1 in human tears, with levels reported at approximately 0.68 ng/100ng tear protein in healthy individuals and 0.35 ng/100ng tear protein in individuals with ocular surface inflammation .

  • ELISA offers high sensitivity and specificity for protein quantification in biological fluids.

• Quantitative PCR (QPCR):

  • QPCR has been used to quantify Slurp1 expression in mouse corneal tissue during different developmental stages and across different mouse strains .

  • This technique is particularly useful for measuring transcript levels when protein detection is challenging.

• Immunofluorescent staining:

  • This method has been employed to examine Slurp1 expression patterns in different mouse strains and age groups .

  • Immunofluorescence provides information about both expression levels and localization within tissues.

• Collection methods for tear samples:

  • Human tears can be collected using absorbent wicks, following appropriate IRB-approved protocols .

  • This non-invasive collection method is suitable for human studies investigating SLURP1 levels.

The choice of method should be determined by the specific research question, sample type, and required sensitivity. For protein level quantification in biological fluids, ELISA is generally recommended, while QPCR is suitable for transcript level analysis in tissue samples.

What cell and animal models are most appropriate for studying SLURP1 function?

Several cell and animal models have proven valuable for investigating SLURP1 function:

• Cell models:

  • Human corneal limbal epithelial (HCLE) cells: These cells can be engineered to express SLURP1 (HCLE-SLURP1) and have been successfully used to study SLURP1's effects on cell cycle progression .

  • E.coli expression systems: These have been used for recombinant production of SLURP1 for structural and functional studies .

• Animal models:

  • Slurp1-null (Slurp1X−/−) mice: These knockout mice provide a valuable model for studying the consequences of SLURP1 deficiency in vivo .

  • Various mouse strains (Balb/C, FVBN, C57Bl/6, and DBA/2J): These have been used to study Slurp1 expression patterns and have shown that expression is consistent across different genetic backgrounds .

• Developmental stages:

  • Studies have examined Slurp1 expression at various developmental stages, including embryonic (E13, E16) and postnatal (PN1, PN10, PN20, PN56) timepoints .

  • This temporal approach is useful for understanding the developmental regulation of SLURP1.

• Experimental manipulation models:

  • Corneal epithelial debridement has been used to study SLURP1's role in wound healing .

  • This model allows for the assessment of wound healing dynamics in the presence or absence of SLURP1.

The choice of model should be guided by the specific aspect of SLURP1 function being investigated. For cellular mechanisms, engineered cell lines offer controlled conditions, while knockout mouse models provide insights into physiological and pathological roles in vivo.

What is the relationship between SLURP1 dysfunction and human diseases?

SLURP1 dysfunction has been implicated in several human pathological conditions:

• Mal de Meleda: SLURP1 gene mutations are linked with this rare autosomal recessive skin disorder . This association highlights SLURP1's critical role in maintaining skin integrity and function.

• Ocular surface inflammation: Tears from inflamed human ocular surfaces contain significantly decreased amounts of SLURP1 (0.35 ng/100ng tear protein) compared with those from healthy individuals (0.68 ng/100ng tear protein) . This reduction suggests that SLURP1 deficiency may contribute to inflammatory processes or serve as a biomarker for inflammation.

• Epithelial barrier dysfunction: Given SLURP1's role in maintaining epithelial integrity through regulation of tight junction and desmosomal proteins, its dysfunction may contribute to conditions characterized by compromised epithelial barriers .

Understanding these disease associations provides opportunities for developing diagnostic tools and therapeutic strategies targeting SLURP1 pathways.

How might SLURP1's cell cycle regulatory properties be exploited in therapeutic applications?

SLURP1's ability to regulate cell cycle progression, particularly its role in stalling G1-S transition, presents several potential therapeutic applications:

• Anti-proliferative therapy: SLURP1's mechanism of downregulating cyclins (CCNE and CCND1/D2), cyclin-dependent kinases (CDK4 and CDK6), and upregulating CDK inhibitor p15/CDKN2B could be exploited in conditions characterized by excessive cell proliferation .

• Tissue engineering and regenerative medicine: SLURP1's pro-differentiation properties could be utilized to promote differentiation of stem cells or progenitor cells in tissue engineering applications.

• Wound healing modulation: Given SLURP1's role in corneal epithelial wound healing, therapeutic modulation of SLURP1 activity might enhance healing in conditions characterized by impaired wound repair .

• Anti-inflammatory applications: SLURP1's anti-inflammatory properties suggest potential utility in treating inflammatory conditions, particularly those affecting epithelial surfaces .

Researchers exploring these therapeutic avenues should consider:

• Delivery methods for SLURP1 or SLURP1-modulating compounds

• Tissue specificity of interventions

• Potential off-target effects given SLURP1's multiple biological functions

• Appropriate dosing to achieve desired cell cycle effects without compromising normal tissue homeostasis