LYVE1 Human

What is the structure and function of human LYVE-1?

Human LYVE-1 is a 322-residue type I integral membrane glycoprotein that functions as a major receptor for hyaluronan (HA) on lymphatic vessel walls. It shares approximately 41% similarity with CD44, another HA receptor, but is uniquely expressed on lymphatic endothelium and absent from blood vessels, making it a powerful lymphatic-specific marker .

Structurally, LYVE-1 contains:

• A 212-residue extracellular domain with a single Link module (the prototypic HA binding domain)

• A transmembrane region

• A cytoplasmic tail

The receptor is located within specialized button-like endothelial junctions of lymphatic vessels and makes the first adhesive contact with incoming immune cells through the formation of endothelial transmigratory cups . Its primary function is mediating the migration of immune cells (particularly dendritic cells and macrophages) through lymphatic vessels, which is critical for immune surveillance and the generation of protective immune responses in draining lymph nodes .

How does LYVE-1 facilitate immune cell trafficking to lymph nodes?

LYVE-1 plays a crucial role in immune cell trafficking through its interaction with hyaluronan (HA) in the surface glycocalyx of tissue-migrating dendritic cells and macrophages. This interaction enables several key processes:

• Initial docking: LYVE-1 facilitates the docking of immune cells with the basolateral surface of initial lymphatic capillaries .

• Transmigration: The LYVE-1- HA interaction enables transmigration of immune cells to the vessel lumen .

• First adhesive contact: Located within specialized button-like endothelial junctions, LYVE-1 makes the first adhesive contact with incoming immune cells through the formation of endothelial transmigratory cups .

The importance of this mechanism has been demonstrated experimentally, as disruption of the LYVE-1- HA axis through gene deletion, monoclonal antibody blockade, or HA depletion significantly impairs the trafficking of antigen-loaded dendritic cells to draining lymph nodes, thereby affecting the priming of antigen-specific T cell responses .

Additionally, diurnal regulation of LYVE-1 gene expression in peripheral lymphatics by the circadian clock system facilitates migration of dendritic cells from tissues to lymph nodes during sleeping hours, when priming of T cell responses is most efficient .

What makes the LYVE-1 binding mechanism to hyaluronan unique?

The binding mechanism of LYVE-1 to hyaluronan (HA) is remarkably distinct from other HA receptors, particularly CD44. Research using dynamic force spectroscopy, crystal structures, and molecular dynamics simulations has revealed several unique properties:

Unusual Sliding Interaction

LYVE-1 binds HA through a sliding interaction where free ends of polymer chains are selectively engaged, clasped, and progressively advanced through a flexible binding groove in the receptor . This is enabled by key conformational rearrangements and lubrication by a cushion of water-mediated hydrogen bonds.

Binding Preference

LYVE-1 binds far more rapidly to the free ends of HA chains than internally (side-on binding), with a preference for non-reducing HA termini. This contrasts with CD44, which displays side-on binding to HA .

Structural Differences

The HA-binding domain (HABD) of LYVE-1 features:

• A deep binding groove with overarching sidechains of residues mLys107/hLys108 and mArg104/hLys105 projecting from the β4/β5 loop

• The loop is braced in position by a disulfide bond (mCys84-Cys105/hCys85-Cys106)

• Key residues mTyr86/hTyr87 and mTrp115/hTrp116 form the lower edge of the groove

This groove is substantially deeper than the analogous structure in CD44, which has a shallower groove and a more open binding surface .

Electrostatic Properties

The HA-binding clefts of human LYVE-1, and to an even greater extent mouse LYVE-1, present a highly concentrated distribution of positive charge, while the relevant surface of CD44 is more neutral .

Water-Mediated Interactions

LYVE-1 features an extensive network of structured waters located above and below the sugar-protein interface, forming numerous indirect hydrogen bonds with the HA chain in dynamic exchange. This large network of structured waters is unprecedented among HA receptors and is thought to contribute to the receptor's unique binding properties .

What are the most effective methods for detecting LYVE-1 in experimental systems?

Researchers have successfully employed multiple techniques to detect LYVE-1 in various experimental systems, each with specific advantages depending on the research question:

Western Blot

For protein-level detection, Western blotting has been effectively used with LYVE-1-specific antibodies. In experimental protocols:

• PVDF membranes are probed with Mouse Anti-Human LYVE-1 Monoclonal Antibody (1 μg/mL)

• Detection is performed using HRP-conjugated Anti-Mouse IgG Secondary Antibody

• LYVE-1 appears as a specific band at approximately 70 kDa under reducing conditions

This approach has been successfully used with various cell lines, including HeLa human cervical epithelial carcinoma, MCF-7 human breast cancer, and 293T human embryonic kidney cell lines .

Flow Cytometry

Flow cytometry provides quantitative analysis of LYVE-1 expression in cell populations:

• Human cells (e.g., HUVEC, PBMC) are stained with Anti-Human LYVE-1 Monoclonal Antibody

• An isotype control antibody serves as a negative control

• Detection employs fluorophore-conjugated secondary antibodies (e.g., Allophycocyanin-conjugated Anti-Mouse IgG)

For specific experiments with human PBMC, cells can be cultured with 50 ng/ml Recombinant Human M-CSF for 10 days before staining .

ELISA

The Human LYVE1 solid-phase sandwich ELISA provides quantitative measurement of LYVE-1:

• Uses a matched antibody pair with a target-specific pre-coated capture antibody

• Can detect LYVE-1 in human serum, plasma, or cell culture medium

• Exclusively recognizes both natural and recombinant human LYVE-1

The assay produces a measurable signal proportional to the concentration of LYVE-1 in the original specimen, allowing for accurate quantification .

Immunohistochemistry

As LYVE-1 is a uniquely powerful marker for lymph vessels, immunohistochemical staining is particularly valuable for visualizing lymphatic vessels in tissue sections. LYVE-1 colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels, making it highly specific for lymphatic endothelium .

How does LYVE-1 dimerization affect its binding properties?

LYVE-1 can form disulfide-linked homodimers in vivo, which significantly impacts its functional properties:

Enhanced Binding Affinity

Dimerization increases LYVE-1's apparent HA binding affinity by approximately 15-fold compared to the monomeric form . This enhanced affinity is likely crucial for its efficient functioning in lymphatic vessel endothelium.

Structural Basis

The critical disulfide-forming cysteine residue responsible for dimerization is C197 in mouse LYVE-1 and C201 in human LYVE-1, located in the juxtamembrane domain . This heavily O-glycosylated region is largely unstructured, which has complicated crystallization attempts of dimeric LYVE-1.

Preserved Binding Mechanics

Despite the significant change in binding affinity, dynamic force spectroscopy analyses have demonstrated that the same unusual HA unbinding mechanics apply to both monomeric and dimeric forms of the receptor . This indicates that the sliding interaction is an intrinsic property of the HA-binding Link domain in LYVE-1, independent of receptor self-association state.

Functional Implications

The significantly increased binding affinity of dimeric LYVE-1 likely enhances its capacity to capture and retain HA-coated immune cells at the lymphatic endothelium, facilitating their subsequent transmigration. This property may be particularly important under physiological conditions where efficient immune cell trafficking is required.

What role does the water-mediated hydrogen bond network play in LYVE-1-HA binding?

The extensive network of structured waters at the LYVE-1-HA interface represents a unique feature among HA receptors and plays several critical roles in the binding interaction:

Dynamic Hydrogen Bonding

The structured waters form numerous indirect hydrogen bonds with the HA chain in dynamic exchange, as revealed by molecular dynamics simulations . This dynamic nature allows for flexibility in the binding interaction while maintaining sufficient binding strength.

Lubrication Effect

These water molecules create a lubricating cushion around the sugar, facilitating the distinctive sliding interaction of HA as it engages with the sugar-binding groove . This "water skating" effect is likely essential for the receptor's ability to progressively advance HA chains through its binding groove.

Enhanced Flexibility

Both the main contact residues and the bound HA chain display a high degree of flexibility in the HA binding cleft, considerably more so than in CD44 . The water-mediated hydrogen bonding network contributes to this flexibility, allowing for conformational adaptations during binding.

Unprecedented Binding Interface

The contribution of such a large network of structured waters to the HA binding interface is unprecedented amongst HA receptors and hyaluronidases . This unique feature distinguishes LYVE-1 from other related receptors and contributes to its specialized function in lymphatic endothelium.

The combination of protein structural dynamics and water-mediated hydrogen bonding in LYVE-1 likely underlies the distinctive sliding interaction with HA, which facilitates its role in mediating immune cell trafficking through lymphatic vessels.

What experimental approaches are most effective for studying LYVE-1-HA interactions?

Several sophisticated experimental approaches have proven effective for investigating the unique properties of LYVE-1-HA interactions:

Dynamic Force Spectroscopy (DFS)

DFS has been instrumental in revealing the physical nature of LYVE-1 binding to HA polymers:

• Demonstrates LYVE-1's preference for binding to free ends of HA chains rather than internal regions

• Shows that HA chains can advance through consecutive LYVE-1 molecules by means of a sliding motion

• Reveals that bound HA can subsequently retract through collective unbinding

This technique is particularly valuable for comparing the binding mechanics of monomeric and dimeric forms of LYVE-1.

X-ray Crystallography

Crystal structures of LYVE-1 HA-binding domains (HABDs) and their HA ligand-bound complexes have provided critical structural insights:

• Revealed the deep binding groove characteristic of LYVE-1

• Identified key residues involved in HA binding

• Elucidated structural differences between mouse and human LYVE-1

• Enabled comparison with other HA receptors like CD44

For crystallography studies, researchers have used soluble extracellular domain constructs of both murine and human LYVE-1, often omitting the heavily glycosylated stalk region that tends to interfere with crystallization.

Molecular Dynamics (MD) Simulations

MD simulations have been crucial for analyzing the dynamics of the binding interaction:

• Revealed the extensive network of structured waters at the binding interface

• Demonstrated the dynamic exchange of water-mediated hydrogen bonds

• Illustrated the flexibility of both the receptor binding groove and the HA ligand

• Provided insights into the mechanics of the sliding interaction

Site-Directed Mutagenesis

This approach has confirmed the involvement of specific residues in HA binding:

• Residues such as mTyr86/hTyr87 and mTrp115/hTrp116, which form the lower edge of the binding groove

• Overarching residues mLys107/hLys108 and mArg104/hLys105

• Disulfide-forming cysteines that brace the critical β4/β5 loop

Sandwich ELISA

For quantitative studies of LYVE-1 levels and binding interactions:

• Employs a target-specific antibody pre-coated in microplate wells

• Utilizes a second detector antibody to form a sandwich with the captured LYVE-1

• Produces a measurable signal proportional to LYVE-1 concentration

• Enables quantitative analysis of LYVE-1 in various biological samples

What are the key structural differences between LYVE-1 and CD44?

Despite their evolutionary relationship and shared function as hyaluronan receptors, LYVE-1 and CD44 exhibit significant structural differences that underlie their distinct binding properties:

Binding Groove Depth

The most striking difference is the deep binding groove in LYVE-1 HABD structures, featuring overarching sidechains of residues mLys107/hLys108 and mArg104/hLys105 projecting from the β4/β5 loop . In contrast, while CD44 has an analogous β4/β5 loop, it is less bulky and overlies a far shallower groove in what is a wider and more open binding surface .

Electrostatic Properties

The HA-binding clefts of human LYVE-1, and to a greater extent mouse LYVE-1, present a highly concentrated distribution of positive charge . In contrast, the relevant surface of CD44 is rather more neutral, and its most basic patch is far removed from the HA binding position .

Binding Coordination

The types of interaction coordinating HA in the binding groove differ significantly:

• LYVE-1 features a greater number of hydrophobic interactions

• LYVE-1 has fewer direct hydrogen bonds compared to CD44

• LYVE-1 utilizes an extensive network of structured waters forming numerous indirect H bonds with the HA chain

Flexibility and Dynamics

Both the main contact residues and the bound HA chain displayed a high degree of flexibility in the LYVE-1 HA binding cleft, considerably more so than in CD44 . This enhanced flexibility contributes to LYVE-1's distinctive sliding interaction with HA.

N-linked Glycosylation

Both receptors feature regulatory N-linked glycans, but their positions and effects differ:

• In LYVE-1, two N-linked glycan sidechains (on mAsn52/159 and hAsn53/160) are associated with in vivo regulation of HA-binding

• These glycosylation sites are specifically positioned to regulate access to the binding groove

These structural differences explain why LYVE-1 has evolved as a key regulator of lymph vessel entry with binding properties distinct from CD44, despite their shared ability to bind hyaluronan.

How does LYVE-1 contribute to inflammation resolution and tissue repair?

Beyond its role in immune cell trafficking, LYVE-1 plays critical functions in inflammation resolution and tissue repair processes:

Macrophage Clearance

LYVE-1 mediates the clearance of macrophages that remove macromolecular debris during the resolution of tissue injury . This process is essential for the restoration of tissue homeostasis following inflammatory responses.

Cardiac Repair

In response to tissue injury, LYVE-1 has been shown to mediate the clearance of inflammatory macrophages from the infarcted heart via epicardial lymphatics . This process is critical for:

• Cardiac repair

• Limitation of subsequent fibrosis

• Prevention of adverse cardiac remodeling

Selective Migration

The unique properties of LYVE-1 binding to HA enable a selective and rapidly reversible mode of interaction, supporting:

• Adherence of immune cells to lymphatic endothelium

• Crawling of these cells along the vessel surface

• Ingress of migrating immune cells from the outer surface of lymphatic capillaries to the lumen

These processes occur in the low shear environment surrounding the lymphatic vasculature, where LYVE-1's specialized binding properties are optimally suited .

Therapeutic Potential

The distinct properties of LYVE-1 suggest it could be exploited for the development of therapies that block unwanted immune and inflammatory responses by disrupting lymphatic trafficking . This approach could be valuable in conditions characterized by excessive immune cell migration or chronic inflammation.

Understanding these additional functions of LYVE-1 highlights its importance beyond basic lymphatic marker status and identifies it as a key regulator of immune homeostasis and tissue repair.

What are the technical considerations for developing LYVE-1-targeted research tools?

Researchers developing tools to study or target LYVE-1 should consider several technical aspects:

Antibody Selection and Validation

When selecting antibodies for LYVE-1 detection:

• Clone #537028 has been validated for Western blot, flow cytometry, and other applications

• Optimal dilutions should be determined by each laboratory for each application

• Validation should include appropriate isotype controls (e.g., MAB002)

• For Western blotting, LYVE-1 appears as a specific band at approximately 70 kDa under reducing conditions

Sample Preparation

Different experimental applications require specific preparation methods:

• For Western blot: Use reducing conditions and appropriate immunoblot buffer (e.g., Buffer Group 1)

• For flow cytometry of human PBMC: Culture with 50 ng/ml Recombinant Human M-CSF for 10 days before staining

• For ELISA: Samples may include human serum, plasma, or cell culture medium

Storage and Handling

For optimal antibody performance:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Storage at -20 to -70°C provides stability for 12 months from date of receipt

• Reconstituted antibodies should be stored according to manufacturer guidelines

Recombinant Protein Design

When designing recombinant LYVE-1 constructs:

• Consider that the extracellular domain spans from Ser24 to Thr238

• The heavily O-glycosylated stalk region (residues 238-290) often interferes with proper folding and crystallization and may be omitted in construct design

• For studying dimerization, include the juxtamembrane domain with the critical disulfide-forming cysteine residue (C197 mLYVE-1 / C201 hLYVE-1)

Assay Validation

For ELISA development:

• Each manufactured lot should be quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency

• The solid-phase sandwich format provides optimal specificity and sensitivity for LYVE-1 detection