Recombinant Nautilus macromphalus Uncharacterized protein IMPP15

What is IMPP15 and why is it significant for research?

IMPP15 is an uncharacterized protein identified in Nautilus macromphalus, a cephalopod mollusk belonging to Nautilidae, one of the few extant cephalopod lineages that retain external biomineralized shells. The significance of IMPP15 stems from its potential role in shell formation processes. Nautilids represent a basally diverging cephalopod group that maintained external shells while most other cephalopods lost or internalized their shells during evolution . Studying IMPP15 could provide insights into the molecular mechanisms of biomineralization and shell evolution in mollusks.

How do shell matrix proteins function in Nautilus species?

Shell matrix proteins in Nautilus species function as essential components in the biomineralization process despite being present in trace amounts within the shell. These proteins play critical roles in calcium carbonate nucleation, regulation of crystal growth, and determination of calcium carbonate polymorphs in the shell structure . The orchestrated activity of various SMPs helps create the highly organized aragonite microstructure characteristic of Nautilus shells. Research indicates that SMPs extracted from Nautilus shells influence crystal growth patterns in vitro, suggesting direct interaction with mineral phases during shell formation.

What methods are typically used to extract and purify native IMPP15 from Nautilus samples?

Native IMPP15 extraction from Nautilus samples typically follows a protocol similar to that used for other shell matrix proteins. The methodology involves:

• Physical separation of the shell by shattering it into pieces

• Cleaning shell fragments with 2M NaOH overnight to remove organic tissues

• Thoroughly washing with ultrapure water (approximately 10 washings)

• Grinding cleaned shell into fine powder

• Slow decalcification using 0.5M EDTA as a chelating agent at 4°C for 72-96 hours

• Extraction of hydrophilic proteins using 3 kDa molecular weight cutoff centrifugal filters

• Storage of extracted proteins at -80°C until further analysis

This approach allows for isolation of the total shell matrix protein fraction, from which individual proteins like IMPP15 can be separated using chromatographic techniques.

What expression systems are most effective for producing recombinant IMPP15?

Multiple expression systems can be utilized for recombinant IMPP15 production, each with specific advantages depending on research requirements. Based on available information, recombinant IMPP15 can be produced in several systems:

The selection of an appropriate expression system should be based on the specific research question, with E. coli being suitable for basic structural studies and mammalian systems preferred when native conformation and post-translational modifications are critical for functional analyses .

How can researchers identify and characterize protein domains in uncharacterized proteins like IMPP15?

Identification and characterization of protein domains in uncharacterized proteins like IMPP15 requires a multi-tool bioinformatics approach. Researchers should:

• Perform sequence analysis using multiple domain prediction tools including:

  • SMART (http://smart.embl-heidelberg.de/)

  • PROSITE (https://prosite.expasy.org/)

  • InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/)

  • NCBI Conserved Domain Database

  • Pfam implemented in HMMER v3.3 (http://hmmer.org/)[1]

• Conduct reciprocal BLAST searches (BLASTp, BLASTx, tBLASTn) against:

  • Non-redundant (nr) protein databases

  • Specialized shell matrix protein databases

  • Genomes of related species

• Compare predicted domains with those found in characterized shell matrix proteins from other mollusks using multiple sequence alignments to identify conserved motifs

• Verify predicted domains through experimental approaches such as limited proteolysis coupled with mass spectrometry to identify domain boundaries

This comprehensive approach has successfully identified protein domains in numerous shell matrix proteins from various molluscan species, including Nautilus pompilius .

What are the optimal sample preparation methods for LC-MS/MS analysis of IMPP15?

Optimal sample preparation for LC-MS/MS analysis of IMPP15 involves several critical steps to ensure high-quality protein identification:

• Protein extraction and purification:

  • Extract total shell proteins as described in method 1.3

  • Further purify using size-exclusion chromatography or affinity chromatography if targeting IMPP15 specifically

• Enzymatic digestion:

  • Reduce disulfide bonds using DTT (typically 10 mM)

  • Alkylate cysteine residues with iodoacetamide (typically 55 mM)

  • Digest with high-quality trypsin (Promega) at a 1:30 enzyme:protein ratio

  • Incubate overnight at 37°C

• Peptide cleanup:

  • Desalt peptide mixtures using C18 spin columns

  • Concentrate peptides by vacuum centrifugation

  • Resuspend in 0.1% formic acid

• LC-MS/MS parameters:

  • Use a nanoLC system (such as DiNa nanoLC)

  • Separate peptides on a C18 analytical column with a gradient of acetonitrile containing 0.1% formic acid

  • Analyze using a high-resolution mass spectrometer (such as LTQ Orbitrap)

• Data analysis:

  • Search spectra against a custom database containing translated transcriptome data

  • Apply appropriate false discovery rate cutoffs (typically 1%)

  • Consider only proteins identified by at least two unique peptides

This methodology maximizes the likelihood of accurate IMPP15 identification and characterization from complex shell matrix protein mixtures.

How does the expression pattern of IMPP15 compare to other shell matrix proteins in the Nautilus mantle?

The expression pattern of IMPP15 in Nautilus macromphalus mantle tissue would need to be analyzed in comparison to other shell matrix proteins using quantitative transcriptomics. While specific data for IMPP15 isn't directly available in the search results, the methodology used for analyzing Nautilus pompilius provides a framework. Comparative expression analysis would typically involve:

• Quantification of transcript abundance using FPKM (Fragments Per Kilobase Million) values derived from RNA-Seq data

• Spatial expression analysis:

  • Extraction of RNA from different regions of the mantle (dorsal vs. ventral regions)

  • Comparison of expression levels between regions directly involved in shell formation and those that are not

• Temporal expression patterns:

  • Analysis of expression during different growth phases or shell repair processes

Based on studies of shell matrix proteins in Nautilus pompilius, shell-specific proteins show highly variable expression levels, with FPKM values ranging from as low as 1.9 to as high as 175,497.3, indicating dramatic differences in transcript abundance . IMPP15 would likely be analyzed within this context to determine its relative expression level and importance in shell formation.

What functional assays can determine the role of IMPP15 in biomineralization?

Several functional assays can elucidate the potential role of IMPP15 in biomineralization processes:

• In vitro crystallization assays:

  • Calcium carbonate precipitation in the presence of purified recombinant IMPP15

  • Analysis of crystal morphology, polymorph selection, and growth kinetics

  • Comparison with crystallization in the absence of IMPP15 or in the presence of known biomineralization proteins

• Calcium-binding assays:

  • Isothermal titration calorimetry (ITC) to measure binding affinity to calcium ions

  • Calcium overlay assays using 45Ca isotopes to visualize calcium-binding properties

• Interaction studies with other shell components:

  • Pull-down assays to identify protein-protein interactions with other shell matrix proteins

  • Surface plasmon resonance to measure binding kinetics with chitin or other organic matrix components

• Localization studies:

  • Immunolocalization using anti-IMPP15 antibodies to determine spatial distribution within the shell

  • In situ hybridization to visualize expression patterns in mantle tissue

• Molecular dynamics simulations:

  • Computational modeling of IMPP15 interactions with calcium carbonate crystal surfaces

  • Prediction of functional domains involved in biomineralization

These methodological approaches would provide comprehensive insights into the functional role of IMPP15 in shell formation and biomineralization processes.

How should researchers address conflicting mass spectrometry data when characterizing IMPP15?

When confronted with conflicting mass spectrometry data during IMPP15 characterization, researchers should implement a systematic troubleshooting and validation approach:

• Sample quality assessment:

  • Evaluate protein purity using SDS-PAGE and western blotting

  • Check for potential contamination from other shell proteins

  • Assess sample degradation through intact mass analysis

• Technical validation:

  • Repeat analyses using multiple LC-MS/MS platforms or conditions

  • Apply different enzymatic digestion strategies (trypsin, chymotrypsin, Lys-C)

  • Use complementary fragmentation techniques (CID, HCD, ETD)

• Data integration approach:

  • Establish minimum identification criteria (e.g., at least two unique peptides with 99% confidence)

  • Create consensus protein sequences from overlapping peptides

  • Map peptides to translated transcriptome data to resolve conflicts

• Sequence validation:

  • Perform de novo sequencing of selected peptides

  • Confirm key peptide sequences using synthetic standards

  • Validate protein sequence using alternative methods such as Edman degradation or targeted MS/MS

• Bioinformatic resolution:

  • Use multiple search engines (SEQUEST, Mascot, MS Amanda) to evaluate confidence in identifications

  • Apply appropriate statistical models for false discovery rate control

  • Consider sequence variants or post-translational modifications that might explain discrepancies

This comprehensive approach minimizes the risk of mischaracterization and ensures reliable identification of IMPP15.

How does IMPP15 compare structurally to characterized shell matrix proteins from other mollusks?

A structural comparison of IMPP15 with characterized shell matrix proteins from other mollusks would involve several analytical approaches:

• Primary structure comparison:

  • Sequence alignment with known shell matrix proteins from other mollusks

  • Identification of conserved motifs and functional domains

  • Analysis of physiochemical properties (hydrophobicity, charge distribution, etc.)

• Domain architecture analysis:

  • Comparison with common domains found in molluscan shell proteins, such as:

    • Carbonic anhydrase domains

    • Chitin-binding domains

    • EGF-like domains (as found in contig_605 in N. pompilius)

    • Sushi domains (as found in contig_171 and contig_8396 in N. pompilius)

• Phylogenetic positioning:

  • Construction of phylogenetic trees to determine evolutionary relationships

  • Analysis of sequence conservation across different molluscan lineages

• Structural prediction and modeling:

  • Secondary structure prediction using algorithms like PSIPRED

  • Tertiary structure modeling using homology modeling or ab initio approaches

  • Comparison of predicted structures with known protein folds

Based on studies of Nautilus pompilius shell matrix proteins, we know that some proteins and domains are conserved across Conchiferans (shelled mollusks), while others appear to be specific to certain lineages . IMPP15, as an uncharacterized protein, would need to be evaluated in this evolutionary context to understand its relationship to other biomineralization proteins.

What evolutionary insights can be gained by comparing IMPP15 with similar proteins in other cephalopods?

Comparative analysis of IMPP15 with similar proteins in other cephalopods can yield significant evolutionary insights:

• Evolutionary trajectory of shell proteins:

  • Comparison with proteins from shell-less cephalopods (octopus, squid) to trace the evolution of shell proteins after shell loss

  • Analysis of whether IMPP15 homologs were repurposed for other functions in shell-less cephalopods

• Selection pressure analysis:

  • Calculation of dN/dS ratios to determine selection pressures on IMPP15 across cephalopod lineages

  • Identification of positively selected sites that might indicate functional adaptations

• Gene family evolution:

  • Determination if IMPP15 belongs to a larger gene family

  • Analysis of gene duplication and diversification events across cephalopod evolution

• Functional domain conservation:

  • Comparison of conserved domains between Nautilus and other cephalopods

  • Assessment of whether specific domains were retained or lost during evolution

• Correlation with morphological evolution:

  • Analysis of how molecular changes in IMPP15 correlate with shell morphology changes in nautiloid evolution

  • Investigation of potential co-evolution with other biomineralization proteins

Such comparative analyses would contribute to understanding the molecular basis of shell evolution in cephalopods, particularly the processes of shell reduction, internalization, or complete loss observed in most extant cephalopod lineages .

What are the most promising research applications for recombinant IMPP15?

Recombinant IMPP15 offers several promising research applications across multiple fields:

• Biomineralization studies:

  • Investigation of protein-mineral interactions in controlled systems

  • Elucidation of molecular mechanisms in shell formation

  • Development of biomimetic mineralization systems

• Evolutionary biology:

  • Functional comparison of IMPP15 with homologs from other mollusks

  • Investigation of the molecular basis for shell diversity

  • Reconstruction of ancestral biomineralization mechanisms

• Structural biology:

  • Determination of three-dimensional structure through X-ray crystallography or cryo-EM

  • Analysis of calcium-binding domains and their conformational changes

  • Study of protein-protein interactions within the shell matrix

• Materials science:

  • Development of bio-inspired materials with controlled crystallization

  • Creation of composite materials with enhanced mechanical properties

  • Design of self-assembling systems based on IMPP15 properties

• Conservation biology:

  • Study of shell formation in threatened nautilus populations

  • Assessment of environmental impacts (ocean acidification, pollution) on shell protein function

  • Development of biomarkers for nautilus population health

These diverse applications highlight the multidisciplinary value of research on recombinant IMPP15 and other shell matrix proteins from basal cephalopods.

How can transcriptomics and proteomics be integrated to better understand IMPP15 function?

Integration of transcriptomics and proteomics provides a powerful multiomics approach to understand IMPP15 function:

• Complementary data generation:

  • RNA extraction and sequencing from mantle tissue regions involved in shell formation

  • Protein extraction and analysis from shell matrix

  • Cross-validation of gene expression and protein abundance

• Integrated analysis workflow:

  • Construction of a species-specific protein database from transcriptome data

  • Use of translated transcriptome sequences to improve protein identification

  • Correlation analysis between transcript abundance and protein detection

• Functional network construction:

  • Identification of co-expressed genes with IMPP15

  • Detection of protein-protein interaction networks

  • Pathway analysis to place IMPP15 in broader biomineralization processes

• Temporal dynamics analysis:

  • Time-course studies of gene expression during shell growth or repair

  • Corresponding analysis of protein incorporation into the shell

  • Identification of regulatory relationships between different shell proteins

• Differential expression studies:

  • Comparison of expression patterns under varied environmental conditions

  • Analysis of protein abundance changes in response to stress factors

  • Correlation with shell morphological or compositional changes

This integrated approach has been successfully applied to Nautilus pompilius, where shell matrix proteins were identified by matching MS/MS spectra to predicted proteins from transcriptome data, resulting in the identification of 61 distinct shell-specific sequences . A similar approach would be highly effective for understanding IMPP15 function.

What experimental designs can test hypotheses about IMPP15's role in shell formation and repair?

Several experimental designs can effectively test hypotheses about IMPP15's role in shell formation and repair:

• In vivo functional studies:

  • RNA interference (RNAi) to knockdown IMPP15 expression in developing Nautilus (challenging but possible in laboratory settings)

  • Monitoring effects on shell formation rate, microstructure, and mechanical properties

  • Rescue experiments with recombinant IMPP15 application

• Ex vivo shell repair models:

  • Creation of controlled damage to Nautilus shells

  • Application of recombinant IMPP15 to repair sites

  • Microscopic and mechanical analysis of repair quality with and without IMPP15

• In vitro biomineralization assays:

  • Design of calcium carbonate crystallization systems with varying concentrations of IMPP15

  • Analysis of crystal nucleation rates, polymorphs, and growth patterns

  • Comparison with crystallization in the presence of other shell proteins

• Structure-function relationship studies:

  • Production of recombinant IMPP15 variants with modified domains

  • Assessment of functional changes in biomineralization capacity

  • Identification of critical residues for mineral interaction

• Environmental response studies:

  • Exposure of Nautilus to varied environmental conditions (pH, temperature, calcium concentration)

  • Analysis of IMPP15 expression changes and shell formation responses

  • Correlation between environmental stress, IMPP15 function, and shell integrity

These experimental designs provide a comprehensive framework for testing specific hypotheses about IMPP15's functional role in shell formation and repair processes, contributing to our understanding of biomineralization mechanisms in Nautilus species.