The Extracellular δ-Domain is Essential for the Formation of CD81 Tetraspanin Webs

Abstract

CD81 is a ubiquitously expressed member of the tetraspanin family. It forms large molecular platforms, so-called tetraspanin webs that play physiological roles in a variety of cellular functions and are involved in viral and parasite infections. We have investigated which part of the CD81 molecule is required for the formation of domains in the cell membranes of T-cells and hepatocytes. Surprisingly, we find that large CD81 platforms assemble via the short extracellular δ-domain, independent from a strong primary partner binding and from weak interactions mediated by palmitoylation. The δ-domain is also essential for the platforms to function during viral entry. We propose that, instead of stable binary interactions, CD81 interactions via the small δ-domain, possibly involving a dimerization step, play the key role in organizing CD81 into large tetraspanin webs and controlling its function.

A mutation in the human tetraspanin CD81 gene is expressed as a truncated protein but does not enable CD19 maturation and cell surface expression

“…This might be due to the low affinity of interaction of the anti-CD81 1D6 with the truncated CD81mut protein or to a diminished affinity of CD81mut for CD19 leading to weak interactions that are not detectable by immunoprecipitation. This reduced association might be due to an unanchored D domain, which is essential for the clustering of CD81 with a different partner, EWI-2, as demonstrated most recently [24].…”

Oligomerization of the Tetraspanin CD81 via the Flexibility of Its δ -Loop

“…The membrane sheets were further stained for sialic acid and N-acetyl-glucosaminyl using wheat germ agglutinin (WGA) conjugated to Alexa Fluor 594 (#W11262; Invitrogen, Waltham, MA) at a concentration of 5 mg/mL in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , pH 7.4) for 1 h at room temperature. After washing, the membrane sheets were imaged using an epifluorescence microscope as described previously (57). The lipid dye 1-(4-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate was added to visualize the membrane, and imaging was performed in the blue (membrane), green (CD81-GFP), and red (WGA-Alexa Fluor 594) channels.…”

“…Jurkat T-cells were cultured and transfected essentially as previously described (57). In brief, 10 5 Jurkat T-cells transfected with a plasmid encoding for CD81-GFP were plated onto poly-lysine-coated coverslips in 1 mL prewarmed Ringer solution (130 mM NaCl, 4 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 48 mM D(þ)glucose, 10 mM HEPES, pH 7.4) for control samples or prewarmed Ringer solution supplemented with 0.5 unit/mL neuraminidase (#N2876; Sigma-Aldrich, St. Louis, MO) and 4 units/mL b 1-4 galactosidase (#P0730; New England Biolabs, Ipswich, MA) for treated samples.…”

The specificity of homomeric clustering of CD81 is mediated by its δ‐loop

“…There are also parallels between biochemistry and microscopy regarding the interaction strength between two partners. For instance, CD81 and its primary interaction partner EWI‐2 overlap stronger with each other than CD81 with its secondary interaction partner CD9 . Moreover, CD53 and CD81 are in closer proximity to their primary interaction partners than to other tetraspanins .…”