L Selectin Human

What is the molecular structure of human L-selectin?

Human L-selectin is a type-I transmembrane glycoprotein composed of 9 exons. It contains an N-terminal calcium-dependent (C-type) lectin domain that interacts with various glycans, including sialyl Lewis X (sLe^X). The human gene includes several splice variants, with L-selectin-v1 and L-selectin-v2 possessing an additional exon between exons 7 and 8. Notably, one human splice variant lacks exon 7, which encodes the transmembrane domain, resulting in a secreted soluble form of L-selectin that circulates in plasma .

How does L-selectin expression differ among leukocyte subtypes?

The regulation of L-selectin expression varies significantly among leukocyte subsets, both at translational and post-translational levels. For example, central memory T-cells express L-selectin differently than neutrophils. Neutrophils demonstrate "basal shedding" of L-selectin, with mouse neutrophils lacking ADAM17 (a sheddase) expressing higher than average surface levels of L-selectin . Each leukocyte subtype has a unique pattern of L-selectin expression that corresponds to its specific function in immune surveillance and inflammatory responses.

What are the differences between membrane-bound and soluble L-selectin?

Soluble L-selectin is detected in the plasma of healthy humans at concentrations of 0.7-1.5 μg per mL, suggesting ongoing cleavage from circulating leukocytes at basal levels . Two mechanisms generate soluble L-selectin: ectodomain shedding by proteinases (primarily ADAM17) and alternative splicing that produces a variant lacking the transmembrane domain. Soluble L-selectin can compete with cell-associated L-selectin, potentially buffering leukocyte recruitment during inflammation, though the precise equilibrium between these forms requires further investigation .

How does L-selectin mediate both rolling interactions and signal transduction?

L-selectin functions beyond its classical role as a tethering/rolling receptor. When engaged through antibody-mediated cross-linking or ligand binding, L-selectin initiates complex signaling cascades involving multiple pathways. These include activation of mitogen-activated protein (MAP) kinases, tyrosine kinases, and the p21 oncoprotein (Ras) . Specifically, p38 MAP kinase becomes phosphorylated within minutes of L-selectin cross-linking, and inhibition of p38 MAP kinase blocks L-selectin-dependent neutrophil shape change, adhesion, and degranulation . These signaling events bridge the gap between initial rolling interactions and subsequent firm adhesion, suggesting L-selectin operates as both an adhesion receptor and a signal transducer during inflammation.

What is the mechanistic basis for L-selectin's role in extravascular migration?

Intravital microscopy studies have revealed that L-selectin significantly influences leukocyte behavior after they leave the vasculature. In L-selectin-deficient mice, leukocytes showed diminished ability to respond to chemotactic stimuli like platelet-activating factor (PAF) and KC (a murine chemokine) . Time-lapse videomicroscopy demonstrated that L-selectin-deficient leukocytes were severely impaired in both random migration (chemokinesis) and directed movement (chemotaxis) within tissues . This suggests L-selectin may interact with extravascular substrates or influence intracellular signaling pathways essential for cytoskeletal reorganization during migration, though the precise mechanisms remain to be fully elucidated.

What flow-based assays are optimal for studying L-selectin function?

Flow chamber assays provide valuable insights into L-selectin function under physiologically relevant shear conditions. A standardized protocol involves:

• Collecting whole blood with acid-citrate dextrose as an anticoagulant

• Diluting blood 1:10 in HBSS and staining leukocytes with rhodamine 6G

• Preparing coverslips with immobilized selectins (e.g., E-selectin)

• Mounting coverslips in a polycarbonate chamber with parallel plate geometry

• Drawing blood through the chamber at defined wall shear stresses (typically 4.0 dynes/cm²)

• Quantifying rolling and adherent cells in multiple random fields

• Performing Wright-Giemsa staining for leukocyte subtype identification

This technique allows for precise quantification of rolling efficiency and firm adhesion, enabling comparative studies between wild-type and L-selectin-deficient leukocytes or testing of inhibitors targeting L-selectin-mediated interactions.

How can researchers effectively isolate and identify L-selectin ligands?

To identify L-selectin ligands, affinity isolation techniques using recombinant selectin fusion proteins are highly effective. For E-selectin ligands:

• Create cell extracts from neutrophils using detergent lysis buffers

• Prepare E-selectin-Ig fusion protein affinity columns

• Apply cell extracts to columns in calcium-containing buffer

• Wash extensively to remove non-specific binding

• Elute bound ligands with EDTA-containing buffer

• Analyze eluates by SDS-PAGE and Western blotting

• Confirm direct binding by reprecipitation assays with purified components

• Validate with sialidase treatment to assess carbohydrate dependency

Notably, this approach revealed species differences, as human L-selectin was identified as a major E-selectin ligand, while mouse L-selectin was not detected in similar experiments .

What in vivo models best represent human L-selectin function?

While mouse models provide valuable insights, researchers should be aware of species-specific differences when studying L-selectin. The cremaster muscle preparation combined with intravital microscopy offers exceptional visualization of leukocyte rolling, adhesion, emigration, and interstitial migration:

• Exteriorize the cremaster muscle in anesthetized mice

• Superfuse with chemotactic inflammatory mediators (PAF or KC)

• Record leukocyte-vessel wall interactions using intravital microscopy

• Quantify rolling, adhesion, and emigration events

• Use time-lapse videomicroscopy to track extravascular migration

For chemotaxis studies, a modified approach using slow release of chemokines from agarose gel positioned at a defined distance (e.g., 350 μm) from postcapillary venules creates directional cues for emigrated leukocytes .

How does L-selectin shedding correlate with inflammatory disease activity?

L-selectin shedding serves as a biomarker for neutrophil activation and inflammatory disease activity. Soluble L-selectin levels are elevated in patients with rheumatic diseases, partially due to increased expression of splice variants lacking the transmembrane domain . The balance between membrane-bound and soluble L-selectin appears to regulate leukocyte recruitment during inflammation, with soluble L-selectin potentially competing for ligands and modulating inflammatory responses. Measuring plasma soluble L-selectin concentrations (normally 0.7-1.5 μg/mL in healthy individuals) can provide insights into disease activity and neutrophil activation status .

How do binding properties differ between human and mouse L-selectin?

A critical species difference exists in L-selectin binding properties. L-selectin from human neutrophils can be affinity-isolated as a major ligand using E-selectin-Ig as an affinity probe, while L-selectin from mouse neutrophils cannot be isolated in similar experiments . This binding of human L-selectin to E-selectin is:

• Direct (purified L-selectin could be reprecipitated with E-selectin-Ig)

• Sialidase-sensitive (abolished by sialidase treatment)

• Calcium-dependent

These findings indicate significant species-specific differences in L-selectin glycosylation patterns or structural features that affect selectin-selectin interactions, complicating the extrapolation of findings between species.

What structural differences account for the functional variations between human and mouse L-selectin?