Worm Breeder's Gazette 13(1): 83 (October 1, 1993)

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.

Molecular Anatomy of the Surface Coat of the Infective L2 of the Parasitic Nematode Toxocara canis (Or: the Sliminess of the Worms).

David Gems, Rick Maizels

Dept. Biology, Imperial College,
Prince Consort Rd., London SW7 2BB UK.

Developmentally arrested L2 s(well, possibly L3 s)of the ascarid parasite T. canis can survive at least 9 years in the host, happily evading the immune system. This facility is believed to be attributable partly to the surface coat: a layer of a 120-kDa glycoprotein ( TES-120 )which overlies the epicuticle, and is continuously shed from the surface of cultured L2 s.

Cloning the Surface Coat Mucin, TES 120

We have demonstrated that TES-120 is almost certainly encoded by a 770 bp hyperabundant mRNA, nmuc1 (nemomucin). The mRNA is trans-spliced with the spliced-leader sequence SL1 and putative nmuc1 protein is 17.6 kDa, increasing in size upon glycosylation. There are three parts: a 16 residue N-terminal hydrophobic signal peptide; an 86 residue mucin-like serine/threonine-rich domain, which contains 72.1% Ser and Thr residues, largely contained in a number of heptamer repeats of the consensus sequence STSSSSA; and a C-terminal cysteine-rich domain.

A Surface-Associated Motif

This latter comprises 2 repeats of a 36 residue sequence containing a consensus XCXD(X)4C(X)6C(X)12C(X)2TC(X)2C, which we call C6 motifs. Comparing the number of residues of the mucin-like domain with size measurements of several mammalian mucins suggests TES-120 to be 17-20 nm long. Since the surface coat is 10-20 nm thick, it looks as if the surface coat comprises a monolayer of TES-120 .Database searching of GenBank and dbEST databases identified 3 C.elegans sequences containing C6 motifs. Two ESTs from mixed stage cDNA libraries, and the ORF zk643 .6on LGIII. The two ESTs, like nmuc1 contain paired C-terminal C6 motifs, and N-terminal domains of unknown length. One EST maps to LGV. zk643 .6comprises five C6 motifs. The nmuc1 mRNA is hyperabundant in L2 s(8.2-13.0% total mRNA!), and carries a 5 SL1 sequence. A second highly abundant cuticular antigen, TES-32 ,contains paired C6 motifs - but at the N-terminus. Given that both TES-32 and TES-120 are surface proteins and have C6 motifs perhaps the C.elegans homologues are also surface proteins?

The TES-32 mRNA comprises 0.8-1.9% of total mRNA, and is also trans-spliced with SL1 .Neither mRNA is expressed in adult Toxocara. The non-C6 part of TES-32 is homologous to a family of phosphatidylethanolamine-binding proteins found in yeast, filarial nematodes and mammals - but nobody knows what the functions of any of these proteins really is. Immuno-EM using a MAB specific to TES-32 localizes it to the outer epicuticle, and it is also secreted in large quantities. This MAB coprecipitates TES-120 ,suggesting that TES-32 may anchor TES-120 to the epicuticle. However, glutaraldehyde cross-linking experiments have not confirmed the hypothetical association of these two proteins.

Surface Coats and the Glycocalyx

The nematode surface coat used to be called the glycocalyx, and a lot of vertebrate epithelial cells also have glycocalyces - electron dense surface layers of highly glycosylated proteins. This applies especially to cells lining ducts (blood vessels, Iymphatics, lungs, gut etc.) Many glycocalyx molecules are small mucins similar to TES-120 (e.g. episialin). Now, some such mucins are overexpressed by tumours - overexpression which is thought to confer immune evasory properties to the tumours. This begs the question: could the mechanisms of mucin-mediated immune evasion by some tumours and some nematodes be one and the same?