Worm Breeder's Gazette 12(1): 51 (September 1, 1991)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Surface-Directed Biotinylation of C. elegans Foiled: An Endogenous Avidin-Binding Protein

Mark Blaxter

Wellcome Centre, Dept. of Biology, Imperial College, London SW7 2BB (while visiting Jim Kramer, Laboratory for Molecular Biology, University of Illinois at Chicago, IL 60686)

Specific labelling of (glyco)proteins on the surface of C. elegans will allow their isolation, characterization and, ultimately, cloning. I reported earlier (WBG 11.2) on the use of lodogen-mediated radio-iodination, and presented further data derived from lodogen studies at the Worm meeting in Madison. Both lodogen (a solid-phase reagent which attacks tyrosine residues) and Bolton-Hunter reagent (a lipophilic iodination reagent attacking lysines) label a small set of proteins on the surface of C. elegans. While labelling efficiencies are an order of magnitude less when compared to parasitic species such as Brugia, they do do the job. One drawback is the need for [125I] and its associated hazards. The biotin-avidin system offers an alternative nonradioactive tag which has been widely used in immunochemistry. Reactive biotin derivatives are available which will attack amino groups (N-hydroxysuccinimidobiotins [NHS-B]) and aldehydes (biotin hydrazide [BHz]). Biotinylated proteins can be analyzed by SDS-PAGE and western blotting with an avidin-peroxidase conjugate. NHS-B has been used to label surface antigens of Brugia and Dirofilaria parasitic nematodes, where it displays similar specificity to lodogen-mediated iodination.

I have tried NHS-B labelling on larval and adult C. elegans N2 using both NHS-B (dissolved in DMF) and a water-soluble sulfo-derivative, SNHS-B (both from Pierce). Labelings (of ~25 adults) were carried out in 10µl of NaHCO3 buffer, pH 8.5, at biotin ester concentrations of 0.1 to 1.0 mg/ml. At room temperature, biotinylation of a large number of products was seen. The profile of products basically followed the Ponceau S staining pattern of the protein content of the worms, indicating that the reagents had penetrated deeply. Reducing the incubation time reduced the intensity of labelling slightly but did not increase specificity. The worms probably swallowed the NHS-B or SNHS-B. The results from labelings carried out on ice were at first encouraging. At all NHS-B and SNHS-B concentrations and at all time points (5 min to 30 min) only a single molecule of 80 kDa appeared to be labelled. It was present in both adults and L1 /L2larvae. However it was also present in unlabeled worm extract. The reactivity seen is not an endogenous peroxidase (no reaction with substrate alone) or a peroxidase-binding protein (it is not seen in ordinary westerns). The 80 kDa band was also present in other free-living nematodes tested (Rhabditis, Oxycerca, Panagrellus, C. briggsae). Two possibilities are: [1] It is a biotinylated protein. The biotin groups would have to be attached by covalent bonds not reduced by 2-ME. [2] It is an avidin-binding protein. Avidin is a 66 kDa tetramer of identical 128 amino acid subunits, each of which carry a single asparagine-linked carbohydrate sidechain. The 80 kDa could recognize either the CHO (and would thus be a "lectin") or the protein (or both). I favor the biotinylated protein model.

BHz will label glycoproteins if the carbohydrates are first derivatised with periodate to produce exposed aldehyde groups. I have not tried this reagent, because [1] 10 mM N aIO4 doesn't sound too good for worms, [2] the reactions should be carried out at room temperature (when the worms will swallow the N aIO4 and the BHz) and [3] the N aIO4 derivatisation works on mammalian glycoproteins by attacking sialic acid residues, but C. elegans doesn't have any surface sialic acid (as determined by radiolectin assays).

Apparent bottom line: Biotinylation isn't an alternative to [125I]. Shame really.

However, the observation that the freeliving nematode surface is refractory to attack by NHS esters is interesting, and distinguishes it from most other biological surfaces. It suggests that [1] there are no exposed amino groups on the surface of C. elegans [or, if there are they aren't on glycoproteins] and [2] the cuticle is highly impermeable. Perhaps worms with more permeable cuticles, a phenotype which might be found in srf mutants or in mutants especially sensitive to SDS, could be extrinsically biotinylated. Such biotinylation might then provide an assay and probe for cuticle permeability and the determinants thereof.