Recombinant Rat Lengsin (Lgsn)

What is rat Lengsin and what is its evolutionary significance?

Rat Lengsin (Lgsn) is a member of the glutamine synthetase (GS) superfamily that exhibits evidence of dynamic evolutionary history in the vertebrate lens. While it shares structural similarities with prokaryotic class I GS proteins, it has undergone significant modifications throughout evolution. The protein shows considerable variability in sequence and length in the N-terminal regions across vertebrate species, while the remainder of the sequence remains well conserved .

Evolutionary analyses have revealed that Lengsin has experienced gain and loss of exons and major changes in the N-terminal domain length. In zebrafish and chicken, the Lengsin gene has four exons, whereas in mammals like rat, it has five exons. This evolutionary divergence suggests that Lengsin may have been re-engineered from an enzymatic protein to serve a structural or binding role specific to the lens .

What is known about the expression pattern of Lengsin in rat lens?

Lengsin shows a remarkably restricted expression pattern in the rat lens. Northern blot analyses of multitissue samples demonstrate that Lengsin mRNA is detectable only in the adult lens, not in retina or in newborn lens tissue . Western blot analyses of dissected epithelial and fiber cells show that Lengsin protein (appearing as a doublet of bands at 62 and 51 kDa) is exclusively present in fiber cells, not in epithelial cells .

In situ hybridization studies reveal that Lengsin is expressed in a ring of fiber cells undergoing terminal differentiation and nuclear loss. This highly specific spatiotemporal expression pattern suggests that Lengsin serves as a marker for terminal differentiation in the lens and may play critical roles in secondary fiber cell morphogenesis and organization .

What is the molecular structure of rat Lengsin protein?

The protein exists in two forms: a full-length 62 kDa form and a truncated 51 kDa species resulting from cleavage at an Asp-Pro sequence near the junction between the N-terminal domain and the remainder of the molecule. This post-translational modification appears to occur both in recombinant protein preparations and in vivo, as detected in lens extracts .

Notable structural differences between Lengsin and bacterial GS include the presence of "pillar" densities on the outer region of the Lengsin dodecamer and differences in the β-hairpin regions that form the central β-sheet collar in GS I dodecamers .

What are the optimal conditions for expressing recombinant rat Lengsin in E. coli?

Based on successful expression protocols for recombinant Lengsin and other rat proteins, the following methodology is recommended:

• Clone the rat Lengsin coding sequence into a pET expression vector system with an N-terminal His-tag for purification purposes.

• Transform the construct into an E. coli expression strain such as BL21(DE3).

• Grow transformed bacteria in LB medium supplemented with appropriate antibiotics at 37°C until mid-log phase (OD600 of 0.6-0.8).

• Induce protein expression with 0.5-1.0 mM IPTG.

• Reduce the temperature to 16-18°C after induction and continue expression overnight to minimize protein aggregation and inclusion body formation.

• Harvest cells by centrifugation and proceed with lysis and purification .

Note that while mouse Lengsin expresses as a soluble protein in E. coli, human Lengsin tends to form insoluble aggregates. Rat Lengsin behavior may vary, requiring optimization of expression conditions to maximize solubility .

What purification strategy should be used for recombinant rat Lengsin?

For efficient purification of rat Lengsin, a multi-step purification process is recommended:

• Cell lysis: Resuspend bacterial pellet in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, and protease inhibitors). Lyse cells using sonication or pressure-based methods.

• Initial purification: Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture the His-tagged Lengsin protein. Use a gradient elution with increasing imidazole concentration (up to 250-300 mM).

• Secondary purification: Apply the eluted protein to size exclusion chromatography (SEC) to separate the dodecameric Lengsin from aggregates and degradation products.

• Quality control: Verify protein purity using SDS-PAGE and Western blotting. Assess the oligomeric state using native PAGE or analytical SEC.

• Storage: Store purified Lengsin at -70°C in a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 10% glycerol to prevent degradation .

Monitor for the appearance of the 51 kDa truncated form during storage, as Lengsin tends to undergo cleavage at the Asp-Pro site near the N-terminal junction .

How should researchers address the N-terminal truncation observed in recombinant rat Lengsin?

The N-terminal truncation of rat Lengsin, resulting in a 51 kDa species from the full-length 62 kDa protein, occurs due to non-enzymatic hydrolysis at an Asp-Pro sequence near the junction between the N-terminal domain and the remainder of the sequence . To address this issue:

• Prevention strategies:

  • Add protease inhibitors during all purification steps

  • Minimize storage time at temperatures above -70°C

  • Consider site-directed mutagenesis to modify the Asp-Pro site without affecting protein function

  • Use freshly purified protein for critical experiments

• Analytical approaches:

  • Perform peptide mass fingerprinting to precisely identify the cleavage site

  • Use N-terminal sequencing to confirm the start of the truncated species

  • Conduct Western blot analysis using antibodies specific to different regions of Lengsin

• Experimental considerations:

  • Compare the functional properties of the full-length and truncated forms

  • Document the ratio of full-length to truncated protein in each preparation

  • Consider separating the two forms using ion exchange chromatography for experiments requiring homogeneous protein

A table summarizing identified peptides from N-terminal truncation analysis:

What methods are appropriate for investigating Lengsin's binding partners in the lens?

Several complementary approaches can be employed to identify and characterize Lengsin's binding partners:

• Yeast Two-Hybrid (Y2H) Screening:

  • Clone full-length rat Lengsin into a bait vector

  • Screen against a lens-specific cDNA library

  • Validate positive interactions with targeted Y2H experiments

  • Note that some fusion proteins (like CP49) may undergo modification in yeast, requiring alternative validation methods

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against rat Lengsin or use epitope-tagged recombinant protein

  • Prepare lens extracts under conditions that preserve protein-protein interactions

  • Immunoprecipitate Lengsin complexes and identify binding partners by mass spectrometry

  • Confirm interactions by reciprocal Co-IP and Western blotting

• Pull-down Assays:

  • Use purified recombinant His-tagged Lengsin as bait

  • Incubate with lens extracts and capture complexes with Ni-NTA beads

  • Analyze bound proteins by SDS-PAGE and mass spectrometry

  • Perform binding assays with purified candidate partners to determine direct interactions

• Proximity Labeling:

  • Express Lengsin fused to BioID or APEX2 in cultured lens cells

  • Allow enzymatic labeling of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

Previous studies have shown that Lengsin can bind to the 2B filament region of vimentin but not to the equivalent regions of CP49 or filensin, suggesting specificity in its interactions with cytoskeletal components .

How can researchers assess the role of Lengsin in lens fiber cell differentiation?

To investigate Lengsin's role in lens fiber cell differentiation, researchers can employ the following methodological approaches:

• Morpholino-mediated Knockdown (validated in zebrafish):

  • Design translation-blocking (targeting start codon) and splice-blocking (targeting exon-intron junctions) morpholinos

  • Inject morpholinos into early embryos

  • Assess lens development through histological analysis and immunostaining

  • Quantify morphological defects in lens fiber organization

• CRISPR-Cas9 Gene Editing:

  • Design guide RNAs targeting early exons of the Lengsin gene

  • Generate knockout rats using embryonic stem cell (ESC)-based methods or direct zygote injection

  • Analyze lens development and transparency in knockout animals

  • Perform rescue experiments with wild-type Lengsin to confirm specificity

• Transgenic Overexpression:

  • Create constructs with Lengsin under control of lens-specific promoters

  • Generate transgenic rats expressing Lengsin at different stages of lens development

  • Examine the effects of altered timing or level of Lengsin expression on fiber cell differentiation

  • Combine with knockout models for rescue experiments

• Ex Vivo Lens Culture:

  • Isolate rat lens at different developmental stages

  • Culture in the presence of Lengsin-targeting siRNAs or expression constructs

  • Monitor fiber cell differentiation through live imaging and endpoint analysis

  • Assess changes in cytoskeletal organization and cell morphology

Statistical analysis of morphological defects should include measurement of lens size, quantification of tissue separations, and assessment of fiber cell organization patterns. In one zebrafish study, the average total lens separation areas were 257 μm² in controls versus 1,175 μm² in Lengsin morphants, demonstrating a statistically significant difference (p<0.05) .

What enzymatic assays have been attempted with recombinant Lengsin, and what are the outcomes?

Despite Lengsin's structural similarity to glutamine synthetase (GS) proteins, extensive enzymatic testing has failed to demonstrate catalytic activity. The following assays have been attempted with recombinant Lengsin:

• Glutamine Synthetase Activity:

  • Forward reaction: glutamate + NH₃ + ATP → glutamine + ADP + Pi

  • Backward reaction: glutamine + ADP + Pi → glutamate + NH₃ + ATP

  • No activity detected with either glutamine or asparagine as substrates

• Alternative Amide Donors:

  • Various potential amide donors, including urea, were tested

  • No nitrogen transfer activity was observed

• Protein Deamidation:

  • Incubation with recombinant crystallins (γS and γD)

  • No change in crystallin charge was observed, indicating no protein deamidase activity

• ATP Binding:

  • Photo-activatable ATP has been used to examine cofactor binding

  • While Lengsin can bind ATP, no catalytic activity has been demonstrated

The lack of enzymatic activity is consistent with sequence analysis showing that critical residues for catalytic activity are not conserved in Lengsin. This suggests that Lengsin may have been evolutionarily repurposed from an enzymatic protein to one with a structural or binding role in the lens, similar to the recruitment of several taxon-specific enzyme crystallins .

How do different rat strains vary in Lengsin expression and function, and how might this impact experimental design?

When designing experiments involving rat Lengsin, researchers should consider strain-specific variations that may affect expression, structure, or function:

• Strain Selection Considerations:

  • Common laboratory rat strains like Sprague-Dawley, Wistar, and Fischer 344 may exhibit differences in Lengsin expression levels or patterns

  • Inbred strains offer genetic homogeneity that reduces experimental variability

  • Outbred strains better represent natural genetic diversity but introduce more variability

• Documented Strain Variations:

  • While specific strain differences in Lengsin have not been thoroughly documented, genetic background can significantly impact lens development and protein expression

  • For example, the DA and ACI rat strains have been successfully used in embryonic stem cell (ESC) studies and may be suitable for Lengsin research

• Experimental Design Recommendations:

  • Maintain consistent strain usage throughout a study

  • Report complete strain information in publications

  • Consider comparing results across multiple strains to assess generalizability

  • For genetically modified rat models, use appropriate background controls from the same strain

  • Account for potential sex differences in Lengsin expression or function

• Quality Control Measures:

  • Verify Lengsin expression levels in each strain before beginning experiments

  • Monitor genetic drift in colony-maintained strains through periodic genotyping

  • Consider using commercially available rats with defined genetic backgrounds for critical experiments

Researchers working with genetically modified rats should be aware that even with CRISPR-based methods, genetic background needs to be controlled to reduce reproducibility problems associated with mixed undefined genomic backgrounds .

What are the challenges and solutions in developing antibodies against rat Lengsin for research applications?

Developing effective antibodies against rat Lengsin presents several challenges that researchers should address with specific strategies:

• Challenge: Cross-reactivity with other GS family members

  • Solution: Design immunizing peptides or recombinant fragments from unique regions of Lengsin, particularly the variable N-terminal domain

  • Approach: Perform sequence alignment of Lengsin with other GS family members to identify Lengsin-specific epitopes

• Challenge: Limited cross-reactivity between species

  • Solution: For species-specific studies, raise antibodies against the corresponding species' Lengsin

  • Evidence: Anti-mouse Lengsin antibodies detect mouse, rat, and rabbit Lengsin, but not human or dog Lengsin

  • Approach: Consider targeting highly conserved regions for cross-species reactivity or variable regions for species specificity

• Challenge: Recognizing both full-length and N-terminally truncated forms

  • Solution: Target epitopes in the conserved C-terminal region present in both forms

  • Approach: Use the purified recombinant protein containing both 62 and 51 kDa species as immunogen

• Challenge: High specificity requirements for immunolocalization

  • Solution: Purify antibodies using affinity chromatography with immobilized recombinant Lengsin

  • Approach: Validate antibody specificity using tissues from Lengsin knockout animals as negative controls

• Challenge: Conformational epitopes in the native dodecameric structure

  • Solution: Immunize with properly folded recombinant protein rather than denatured protein or peptides

  • Approach: Use native protein for immunization and develop conformational epitope-specific antibodies

Recommended protocol for antibody development:

• Express and purify soluble recombinant rat Lengsin as described in section 2.2

• Immunize rabbits or other suitable hosts with the purified protein

• Collect antisera and test reactivity in Western blots of lens extracts

• Validate antibody specificity through immunohistochemistry on rat lens sections

• If necessary, perform affinity purification using recombinant Lengsin

This approach has successfully generated polyclonal antisera that detect both the 62 and 51 kDa Lengsin species in lens extracts .

How can researchers effectively analyze the complex dodecameric structure of rat Lengsin and its relationship to function?

The dodecameric structure of rat Lengsin presents analytical challenges that require specialized approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Methodology: Prepare purified Lengsin at 0.5-2.0 mg/ml in a buffer suitable for vitrification

  • Analysis: Perform single-particle reconstruction to achieve high-resolution structural information

  • Advantage: Allows visualization of the native dodecameric structure without crystallization

  • Challenge: Requires high-quality, homogeneous protein preparations

  • Resolution enhancement: Use of direct electron detectors and advanced image processing algorithms can improve resolution

• Comparative Structural Analysis:

  • Methodology: Compare Lengsin structure with bacterial GS structures through rigid body refinement

  • Key findings: Lengsin has additional "pillar" densities not present in bacterial GS

  • Sequence analysis: Examine regions like the loop sequence between positions 430-435 (in GS numbering) that may differ between Lengsin and GS

• Structure-Function Analysis:

  • Site-directed mutagenesis: Target residues in regions that differ from bacterial GS

  • Domain swapping: Create chimeric proteins between Lengsin and bacterial GS to identify functional domains

  • Truncation analysis: Compare properties of full-length and N-terminally truncated forms

• Protein-Protein Interaction Mapping:

  • Cross-linking mass spectrometry: Identify residues involved in inter-subunit contacts

  • Hydrogen-deuterium exchange: Map surface accessibility and conformational dynamics

  • Computational docking: Model interactions with binding partners like vimentin

• Stability and Assembly Analysis:

  • Analytical ultracentrifugation: Determine oligomeric state and assembly kinetics

  • Differential scanning calorimetry: Assess thermal stability of the dodecameric complex

  • Native mass spectrometry: Analyze intact complexes and subcomplexes

Image reconstruction has revealed that Lengsin forms a structure with dimensions of 140 Å in diameter and ~100 Å in height, with a calculated molecular weight of 744,372 Da for the full-length dodecamer or 605,952 Da for the N-terminally truncated form .

How can recombinant rat Lengsin be used as a marker for terminal differentiation in lens research?

Recombinant rat Lengsin offers several valuable applications as a marker for terminal differentiation in lens research:

• Antibody-based Detection Systems:

  • Immunohistochemistry: Using anti-Lengsin antibodies to identify terminally differentiating fiber cells in tissue sections

  • Immunofluorescence: Performing co-localization studies with other differentiation markers

  • Flow cytometry: Sorting lens cell populations based on Lengsin expression

  • Western blotting: Quantifying Lengsin expression levels during differentiation processes

• Transgenic Reporter Systems:

  • Lengsin promoter-driven reporters: The 3 kb genomic fragment upstream of Lengsin can drive expression of reporter genes like EGFP specifically in terminally differentiating lens fibers

  • Applications: Tracking differentiation in vivo, isolating specific cell populations, testing effects of experimental manipulations on differentiation

  • Validation: Transgenic lines using Lengsin promoter-EGFP constructs recapitulate the temporal and spatial expression patterns of endogenous Lengsin

• Developmental Stage Marker:

  • Timeline reference: Lengsin expression follows the temporal expression of αA-, αB1-, and βB1-crystallin proteins in the lens

  • Spatial reference: At 72 hpf in zebrafish, Lengsin localizes to a subpopulation of differentiating secondary fiber cells, while no expression is detected in epithelial cells or central lens fibers

  • Application: Using Lengsin expression to standardize developmental staging across experiments

• Quality Control for Differentiation:

  • In vitro differentiation: Monitoring successful lens fiber differentiation in culture systems

  • Tissue engineering: Verifying proper terminal differentiation in engineered lens tissues

  • Stem cell research: Confirming lens lineage commitment in differentiation protocols

For developmental studies, researchers should note that Lengsin expression is first detected in zebrafish at 24 hours post-fertilization (hpf), with protein detectable by immunostaining at 30 hpf. In adult lenses, Lengsin is restricted to a narrow band of cortical fibers and co-localizes with actin at the lateral faces of interdigitating cells .

What are the implications of Lengsin's interaction with vimentin for lens cytoskeletal research?

The demonstrated interaction between Lengsin and vimentin offers significant insights for lens cytoskeletal research:

• Mechanistic Implications:

  • Lengsin binds specifically to the 2B filament region of vimentin but not to equivalent regions in CP49 or filensin, suggesting selective cytoskeletal interactions

  • This interaction may help organize the cytoskeleton during terminal differentiation of lens fiber cells

  • The dodecameric structure of Lengsin could potentially cross-link multiple vimentin filaments, contributing to the specialized cytoskeletal architecture of lens fibers

• Research Approaches:

  • In vitro filament assembly: Assess how recombinant Lengsin affects vimentin polymerization and network formation

  • FRET or BRET analysis: Measure direct interactions in living cells

  • Super-resolution microscopy: Visualize co-localization patterns at nanoscale resolution

  • Electron microscopy: Examine ultrastructural relationships between Lengsin and vimentin filaments

• Functional Studies:

  • Compare cytoskeletal organization in normal and Lengsin-deficient lenses

  • Examine whether Lengsin-vimentin interactions are regulated by post-translational modifications

  • Investigate if the N-terminal truncation of Lengsin affects its interaction with vimentin

  • Determine whether Lengsin-vimentin interactions change during lens aging or cataract formation

• Experimental Design Considerations:

  • Use both full-length and N-terminally truncated Lengsin in binding studies

  • Consider species differences in vimentin sequence when designing experiments

  • Account for potential regulatory factors in the cellular environment that may modify interactions

  • Develop methods to quantify binding affinities and kinetics under physiological conditions

This interaction may explain the observed morphological defects in Lengsin knockdown models, where disruption of proper cytoskeletal organization could lead to the observed separations within the lens cortex and defects in secondary fiber morphogenesis .

How can researchers integrate Lengsin studies with investigations of other lens-specific proteins to understand terminal differentiation?

Integrating Lengsin research with studies of other lens-specific proteins requires a comprehensive, multi-layered approach:

• Coordinated Expression Analysis:

  • Temporal profiling: Compare expression timelines of Lengsin with crystallins, intermediate filament proteins, and other lens-specific factors during development

  • Spatial mapping: Determine precise localization patterns of multiple proteins within the lens using multiplexed immunostaining or in situ hybridization

  • Single-cell transcriptomics: Profile gene expression at single-cell resolution to identify co-regulated gene clusters during differentiation

• Protein Interaction Networks:

  • Interactome mapping: Perform systematic protein-protein interaction screens to identify links between Lengsin and other lens proteins

  • Co-immunoprecipitation studies: Isolate multi-protein complexes containing Lengsin from lens tissue

  • Proximity labeling: Use BioID or APEX2 fusions to identify proteins in close proximity to Lengsin in vivo

  • Network analysis: Construct interaction maps to identify key hubs and regulatory connections

• Functional Genomics Approaches:

  • Combinatorial knockdowns/knockouts: Assess synergistic effects of modulating Lengsin together with other lens proteins

  • Transgenic models: Create rats expressing reporters for multiple lens proteins to track differentiation dynamics

  • CRISPR screens: Perform genome-wide or targeted screens to identify factors affecting Lengsin expression or function

• Integrative Analysis Tools:

  • Pathway analysis: Identify signaling pathways connecting Lengsin with other differentiation factors

  • Gene Ontology enrichment: Categorize functional relationships among co-expressed genes

  • Proteomics data integration: Combine protein expression, modification, and interaction data

  • Comparative genomics: Examine evolutionary conservation of lens differentiation programs

• Experimental Systems:

  • Lens-specific promoters: The γD-crystallin promoter contains activating sequences active in lens cells and silencing sequences active in non-lens cells that could be used in conjunction with Lengsin regulatory elements

  • Transgenic resources: The Lengsin promoter (3 kb upstream fragment) has been validated to drive lens-specific expression and can be used to create multi-reporter systems

  • Differentiation models: Compare Lengsin with factors like FGF2, which promotes retinal lens fiber differentiation through ERK1/2 phosphorylation

For example, researchers could create a transgenic rat model with Lengsin promoter driving one fluorescent reporter and crystallin promoters driving different colored reporters to visualize the progression of differentiation in real-time and in fixed tissue samples.