Recombinant Human Low-density lipoprotein receptor-related protein 10 (LRP10), partial

What is LRP10 and how is it structurally characterized?

LRP10 (Low-density lipoprotein receptor-related protein 10) belongs to a distinct subfamily of LDL receptor proteins that includes LRP3 and LRP12. The mature human LRP10 consists of a 424 amino acid extracellular domain (ECD), a 21 amino acid transmembrane segment, and a 252 amino acid cytoplasmic domain. The ECD contains 4 LDLR-A domains and 2 CUB (C1r/C1s, Uegf, Bmp1) domains . Within the ECD, human LRP10 shares 90% amino acid sequence identity with mouse and rat LRP10 .

Unlike classical LDL receptor family members, LRP10 has smaller extracellular regions, which makes it structurally unique, suggesting specialized functions beyond typical lipoprotein metabolism .

What is the cellular localization and tissue distribution of LRP10?

LRP10 is predominantly localized in the trans-Golgi network (TGN), plasma membrane, retromer, and early endosomes, suggesting its role in intracellular trafficking . In human brain tissue, LRP10 is predominantly expressed in astrocytes and neurovasculature but is undetectable in neurons under normal conditions . Similarly, in studies using induced pluripotent stem cell (iPSC) models, LRP10 shows high expression in iPSC-derived astrocytes but cannot be observed in iPSC-derived neurons . This cell type-specific expression pattern suggests that LRP10-mediated pathology may occur through non-neuronal mechanisms in neurological diseases.

LRP10 is also expressed in various other tissues and may be involved in apolipoprotein internalization , indicating its potential role in lipid metabolism across different organ systems.

How is LRP10 involved in intracellular trafficking?

LRP10 functions as a trafficking protein that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes . Its cytoplasmic domain contains acidic dileucine (DXXLL) motifs that are crucial for its trafficking function . In vesicle trafficking pathways, LRP10 partially co-localizes and interacts with sortilin-related receptor 1 (SORL1) , suggesting coordination between these receptors in protein transport.

Experimental approaches to study LRP10 trafficking typically involve:

• Fluorescent tagging of LRP10 for live-cell imaging

• Co-localization studies with organelle markers

• Immunoprecipitation to identify trafficking partners

• Mutational analysis of trafficking motifs in the cytoplasmic domain

In patients carrying the LRP10 p.Arg235Cys variant, significantly enlarged LRP10-positive vesicles have been detected , indicating disrupted trafficking that may contribute to disease pathogenesis.

What is the role of LRP10 in neurodegenerative diseases?

Loss-of-function variants in the LRP10 gene have been associated with:

• Autosomal-dominant Parkinson's disease (PD)

• PD dementia

• Dementia with Lewy bodies (DLB)

• Progressive supranuclear palsy

• Amyotrophic lateral sclerosis

LRP10 has been detected in Lewy bodies (LB) at late maturation stages in brains from idiopathic PD and DLB patients, as well as in LRP10 variant carriers . The high expression of LRP10 in non-neuronal cells, coupled with its undetectable levels in neurons under normal conditions, suggests that LRP10-mediated pathogenicity may be initiated via cell non-autonomous mechanisms .

Key LRP10 variants associated with neurodegenerative conditions include:

All patients carrying these variants had a positive family history for dementia or PD , supporting the genetic link between LRP10 and neurodegenerative conditions.

How can researchers effectively study LRP10-protein interactions?

Several methodological approaches have been validated for studying LRP10 interactions:

• Co-immunoprecipitation (Co-IP):

  • Effective for identifying direct binding partners of LRP10

  • Example protocol: Cells expressing LRP10 constructs can be lysed in RIPA buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Na-Deoxycholate) with protease inhibitors (1 mM PMSF, 1 μg/ml Aprotinin)

  • Protein complexes can be precipitated using antibodies against LRP10 or potential interaction partners

• GST pull-down assays:

  • Useful for confirming direct interactions

  • For example, in-vitro translated LRP10 has been shown to bind strongly to GST-APP-N-terminus and weakly to GST-APP-C-terminus

• Proximity-based labeling methods:

  • BioID or APEX techniques for identifying proximal proteins in living cells

• Confocal microscopy for co-localization studies:

  • Can be performed using the Leica SP5 AOBS confocal microscope with appropriate lasers (diode 405, OPSL 488, DPSS 561, and HeNe 633)

  • Images can be analyzed using Fiji/ImageJ software with the coloc2 tool to determine Manders' overlap coefficient

When examining LRP10 interactors, it's important to consider both the extracellular domain interactions and the cytoplasmic domain interactions, as they may mediate different functions.

What approaches are recommended for studying LRP10 in cellular models?

Several effective approaches have been validated in the literature:

• LRP10 overexpression systems:

  • Plasmid constructs such as pLVX-EF1α-LRP10-IRES-NeoR for stable expression

  • Doxycycline-inducible expression systems (pCW57-LRP10-2A-PuroR) for controlled expression

• Gene knockout strategies:

  • CRISPR-Cas9 system using pSpCas9-GFP-LRP10gRNA or pSpCas9-PuroR-LRP10gRNA plasmids

  • Example: LRP10 knockout mice (Lrp10−/−) have been generated and show viable, fertile phenotypes with specific immune system alterations

• Domain-specific mutants:

  • Expression of LRP10-ECD (extracellular domain only) to study domain-specific functions

  • Splice variants such as LRP10-Splice for modeling disease-associated variants

• iPSC-derived cell models:

  • Patient-derived iPSCs carrying LRP10 variants can be differentiated into relevant cell types (astrocytes, neurons) to study disease mechanisms

Statistical analysis of experiments should employ appropriate tests based on data distribution. For non-normally distributed data, the non-parametric Mann-Whitney U test is recommended .

How can recombinant LRP10 be utilized in binding studies?

Recombinant human LRP10 is commercially available as an Fc chimera protein containing the extracellular domain (His17-Pro438) . This construct can be used for:

• Binding kinetics analysis:

  • Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) to measure association and dissociation rates with potential ligands

  • Example: Biotinylated-LRP1 domains immobilized on Octet streptavidin-biosensors were used to study interactions with ricin toxin

• Pulldown assays:

  • Recombinant LRP10-Fc can be immobilized on protein A/G beads to capture binding partners from cellular lysates

  • Elution and mass spectrometry analysis can identify novel interactors

• Competitive binding assays:

  • To identify compounds that can disrupt pathological interactions

• Cell-based binding assays:

  • Flow cytometry using labeled recombinant LRP10 to identify cell types with binding capacity

When designing binding studies, researchers should consider the physiological pH and ionic conditions, as these may significantly affect LRP10 interactions.

How does LRP10 influence amyloid precursor protein trafficking and processing?

LRP10 has been identified as a regulator of amyloid precursor protein (APP) trafficking and processing, which has implications for Alzheimer's disease research:

• Direct interaction with APP:

  • LRP10 interacts directly and predominantly with the ectodomain of APP in vitro

  • This interaction can be studied using co-immunoprecipitation and GST pull-down assays

• Effect on APP processing:

  • LRP10 modulates APP trafficking within the secretory and endocytic pathways

  • This affects the interaction of APP with β- and γ-secretases and subsequent Aβ production

• Methodological approach for studying this relationship:

  • Express LRP10 and APP in cellular models and examine APP localization

  • Measure Aβ production using ELISAs for Aβ40 and Aβ42

  • Track APP trafficking using fluorescent tagging and live-cell imaging

  • Analyze the effect of LRP10 variants on APP processing

This research area is particularly relevant for understanding the potential role of LRP10 in Alzheimer's disease pathogenesis and developing therapeutic strategies targeting LRP10-APP interactions.

How does LRP10 regulate immune cell homeostasis?

Recent research has revealed an unexpected role for LRP10 in immune regulation, particularly in CD8 T cell homeostasis:

• Effect on CD8 T cell populations:

  • Deletion of Lrp10 in mice causes accumulation of naïve and central memory CD8 T cells in peripheral lymphoid organs

  • Lrp10−/− mice show an increased proportion of CD8 T cells and a reduction in NK cells in peripheral blood

• Mechanism of immune regulation:

  • LRP10 is induced with T cell activation

  • LRP10 post-translationally suppresses IL7 receptor (IL7R) levels

  • LRP10 binds to IL7R through its extracellular domain (ECD) and uses its intracellular domain (ICD) to impair IL7R glycosylation and maturation

• Impact on anti-tumor immunity:

  • Lrp10-deficient mice are intrinsically resistant to syngeneic tumors

  • This resistance depends on dense tumor infiltration of CD8 T cells with increased memory characteristics and reduced terminal exhaustion

These findings position LRP10 as a negative regulator of CD8 T cell homeostasis and a host factor that controls tumor resistance, with potential implications for immunotherapy development.

What experimental designs are recommended for studying LRP10 in immune function?

Based on published methodologies, researchers interested in LRP10's immune function should consider:

• Bone marrow chimera studies:

  • Lethally irradiated Rag2−/− mice reconstituted with Lrp10−/− or Lrp10+/+ bone marrow can determine whether immune phenotypes are hematopoietic-intrinsic

• Homeostatic expansion assays:

  • Co-transfer of labeled Lrp10+/+ and Lrp10−/− T cells into lymphopenic hosts

  • Analysis of cellular proliferation using dye dilution (e.g., Cell Trace Violet)

• Cytokine receptor signaling analysis:

  • Examination of IL7R signaling and downstream pathway activation

  • Western blot analysis of signaling molecules including STAT5 phosphorylation

• Glycosylation analysis:

  • Treatment with deglycosylases (e.g., PNGase) to examine glycosylation states of receptors

  • Western blot to assess molecular weight shifts indicating differential glycosylation

• Tumor resistance models:

  • Challenge with syngeneic tumor cell lines

  • Analysis of tumor-infiltrating lymphocytes

  • Response to immune checkpoint inhibition

These methodological approaches provide a comprehensive framework for investigating the role of LRP10 in immune function across different experimental contexts.

How should factorial experimental designs be implemented when studying LRP10 function?

When investigating complex interactions involving LRP10, proper experimental design is crucial:

Implementing these experimental design principles will maximize the efficiency and statistical power of LRP10 research projects.

What are the key considerations for studying disease-associated LRP10 variants?

When investigating LRP10 variants associated with neurodegenerative diseases, several methodological considerations are important:

• Model system selection:

  • Patient-derived iPSCs differentiated into relevant cell types

  • CRISPR-engineered cell lines with specific variants

  • Transgenic animal models expressing human LRP10 variants

• Analysis of variant effects on LRP10 function:

  • Trafficking studies using fluorescent-tagged constructs

  • Protein-protein interaction changes using co-immunoprecipitation

  • Secretion patterns (e.g., aberrant high molecular weight species in LRP10-Splice)

• Protein analysis techniques:

  • Western blotting with non-ionic (1% NP-40) versus ionic (1% SDS) detergent extraction to reveal different aspects of protein behavior

  • Mass spectrometry to identify post-translational modifications

  • Immunohistochemistry in patient tissue samples

• Mechanism studies:

  • Dominant negative effects (e.g., LRP10-Splice binding to wild-type LRP10 and reducing its levels)

  • Effects on α-synuclein levels and distribution in Lewy body disease models

• Statistical approaches:

  • Power calculations to determine appropriate sample sizes

  • Multiple comparison corrections when testing several variants

  • Consideration of biological versus technical replicates

These considerations will help researchers design rigorous studies that provide meaningful insights into how LRP10 variants contribute to disease pathogenesis.