Recombinant Human Inter-alpha-trypsin inhibitor heavy chain H6 (ITIH6), partial

What is ITIH6 and how does it relate to other members of the inter-alpha-trypsin inhibitor family?

ITIH6 (Inter-alpha-trypsin inhibitor heavy chain H6) belongs to the inter-alpha trypsin inhibitor heavy chain (ITIH) family. Unlike other ITIH family members, ITIH6 is located on chromosome X at position p11.22 (Xp11.22), while most other ITIH proteins are encoded by autosomal genes . The ITIH family is involved in stabilization of the extracellular matrix (ECM), with the heavy chains mediating protein-protein interactions with ECM components .

ITIH6 is also known by several aliases:

• Inter-alpha inhibitor H5-like protein (ITIH5L)

• Inter-alpha-trypsin inhibitor heavy chain H5-like protein

• UNQ6369/PRO21074

• dJ14O9.1

The protein has an Entrez Gene ID of 347365 and UniProt ID of Q6UXX5 (Human) .

What is the genomic organization of the ITIH6 gene?

The ITIH6 gene consists of 13 exons spanning approximately 49,338 base pairs on the complementary strand of chromosome X . The genomic reference sequence is NG_013240.1, while the transcript reference is NM_198510.2 . This genomic organization is important for designing primers for gene amplification and expression studies.

How conserved is ITIH6 across species?

Sequence analysis reveals moderate conservation of ITIH6 across mammalian species. The recombinant ITIH6 control fragment (aa 478-596) shows highest antigen sequence identity to mouse and rat orthologs at only 27% . This relatively low conservation suggests potential functional divergence across species and highlights the importance of species-specific experimental approaches when studying ITIH6.

What are the recommended experimental approaches to study ITIH6 function?

When designing experiments to study ITIH6 function, researchers should follow these methodological steps:

• Define clear research questions: Determine specific aspects of ITIH6 function to investigate (e.g., protein-protein interactions, role in ECM stabilization)

• Select appropriate experimental systems:

  • Cell-based assays using relevant cell types (e.g., liver cells, as ITIH proteins are primarily produced in liver)

  • Recombinant protein studies for biochemical characterization

  • Animal models for in vivo functional studies (consider the 27% homology with mouse/rat when interpreting results)

• Incorporate controls:

  • Use recombinant ITIH6 control fragments for antibody validation

  • Include other ITIH family members to distinguish ITIH6-specific functions

  • Employ negative controls lacking ITIH6 expression

• Validate findings through multiple approaches:

  • Combine biochemical, cell-based, and if possible, in vivo approaches

  • Use both gain-of-function and loss-of-function experimental designs

  • Consider between-subjects or within-subjects designs depending on your research question

How should researchers design antibody validation experiments using recombinant ITIH6?

For proper antibody validation using recombinant ITIH6 control fragments, follow this methodology:

• Pre-incubation protocol:

  • Use a 100x molar excess of the protein fragment control based on antibody concentration and molecular weight

  • Pre-incubate the antibody-protein control fragment mixture for 30 minutes at room temperature

• Blocking experiment design:

  • Prepare parallel experiments with and without pre-incubation with the recombinant ITIH6 fragment

  • Include gradients of recombinant protein concentration to establish dose-dependency

  • Document signal reduction in Western blot, IHC, or ICC applications as evidence of antibody specificity

• Controls and validation:

  • Include positive controls (known ITIH6-expressing samples)

  • Use negative controls (tissues or cells not expressing ITIH6)

  • Compare results with at least one alternative antibody targeting a different epitope of ITIH6

What mass spectrometry approaches are recommended for characterizing ITIH6 glycosylation patterns?

Based on methodologies used for other ITIH family proteins, researchers should consider the following approach for ITIH6 glycosylation analysis:

• Sample preparation:

  • Purify recombinant or serum-derived ITIH6

  • Perform tryptic/GluC digestion to generate glycopeptides

  • Enrich glycopeptides using hydrophilic interaction liquid chromatography (HILIC)

• Glycosylation site confirmation:

  • Treat with PNGaseF in the presence of ¹⁸O water to confirm N-glycosylation sites through mass shift analysis

  • Look for both consensus (N-X-S/T) and potential non-consensus glycosylation sites, as found in other ITIH proteins

• Characterization of glycoforms:

  • Perform glycosidase-assisted LC-MS/MS analysis

  • Analyze site-specific microheterogeneity of N-glycoforms

  • Document both N- and O-glycosylation sites and their occupancy

By comparing recombinant ITIH6 produced in different cell lines with serum-derived ITIH6, researchers can identify cell-type specific glycosylation patterns that may affect protein function.

How can researchers address challenges in reproducing ITIH6-related experimental results?

Reproducibility challenges in ITIH6 research can be addressed by implementing these methodological approaches:

• Standardize recombinant protein production:

  • Use consistent expression systems (e.g., HEK293 cells as used for other ITIH proteins)

  • Document complete details of expression constructs, including any variations from canonical sequences

  • Characterize post-translational modifications of recombinant proteins

• Implement robust experimental design principles:

  • Use appropriate sample sizes based on power calculations

  • Pre-register experimental protocols and analysis plans

  • Include both technical and biological replicates

  • Implement randomization and blinding where applicable

• Comprehensive reporting:

  • Document all experimental conditions in detail

  • Report both positive and negative results

  • Share raw data and analysis scripts through repositories

  • Clearly state limitations of experimental approaches

What approaches are recommended for studying ITIH6 genetic variants?

When investigating genetic variants in ITIH6, researchers should employ these methodological steps:

• Variant identification:

  • Access existing variant databases like LOVD (Leiden Open Variation Database), which contains 48 unique public DNA variants for ITIH6

  • Consider whole-exome sequencing for novel variant discovery

  • Use targeted sequencing approaches for focused analysis of the ITIH6 locus

• Functional characterization:

  • Implement recessive allele frequency-based testing (RAFT) for rare variant analysis, which has higher power than conventional tests for rare recessive variants (<5% allele frequency)

  • Employ stringent filtering and quality control steps to remove false positives from sequencing data

  • Control for population stratification effects that can result in excess homozygosity

• Variant interpretation:

  • Assess conservation across species (considering the 27% homology with mouse/rat)

  • Use prediction tools to evaluate potential functional impacts

  • Perform in vitro functional assays to validate computational predictions

How can researchers design experiments to investigate potential roles of ITIH6 in complex diseases?

Based on methodologies used for other genes in complex disease research, consider the following experimental design approach:

• Hypothesis formulation:

  • Develop specific, testable hypotheses about ITIH6's role in disease pathways

  • Consider both recessive and dominant genetic models

  • Focus on biochemical pathways involving extracellular matrix interactions

• Case-control association studies:

  • Compare rates of rare homozygous or compound heterozygous variants in ITIH6 between cases and controls

  • Consider sample sizes of at least 1,000 cases and 1,000 controls based on previous studies of rare variants

  • Implement appropriate statistical methods for rare variant analysis

• Functional validation:

  • Develop cell-based assays to test the impact of identified variants

  • Consider adaptation of biochemical assays similar to those used for other genes like DHCR24

  • Use animal models when appropriate, while considering the limited homology between human and mouse ITIH6

What are the optimal conditions for storing and handling recombinant ITIH6?

Based on protocols for similar recombinant proteins, researchers should consider these guidelines:

• Storage recommendations:

  • Store at temperatures ≤ -70°C for long-term stability

  • Aliquot the protein to avoid repeated freeze-thaw cycles

  • Store in appropriate buffer (typically PBS) with possible addition of stabilizing agents

• Handling precautions:

  • Thaw recombinant proteins on ice when preparing for experiments

  • Centrifuge briefly after thawing to collect contents at the bottom of the tube

  • Use appropriate personal protective equipment as with all biological materials

  • Remember that recombinant proteins are for research use only, not for diagnostic procedures or human administration

• Quality control:

  • Periodically assess protein integrity using SDS-PAGE

  • Verify activity using functional assays relevant to ITIH6

  • Document lot-to-lot variation when using recombinant proteins in longitudinal studies

How can the functionality of recombinant ITIH6 be verified before experimental use?

To ensure recombinant ITIH6 retains its functionality for experiments, implement these verification steps:

• Biochemical characterization:

  • Verify protein purity (≥90%) by SDS-PAGE

  • Confirm molecular weight and integrity by western blotting

  • Assess immunoreactivity with specific antibodies

• Functional assays:

  • Design binding assays to verify interaction with known partners

  • Develop activity assays based on predicted function in extracellular matrix stabilization

  • Confirm appropriate inhibitory activity if applicable, using methods similar to those for other IAIP family members

These methodological approaches will help ensure reliable and reproducible results when working with recombinant ITIH6 in research applications.