By: Richard D. Cummings
One of the most unusual glycoproteins and one used commonly in biological research and immunology is KLH, which is derived from the giant keyhole limpet (Megathura crenulata). The keyhole limpet is a giant aquatic snail (gastropod) with a cone-shaped shell (shell length can be 13 centimeters). [Its name derives from the tiny hole at the tip of its shell.] The keyhole limpet is non-edible and resides in the Pacific coastal waters off California and Mexico. It lacks typical blood and has no red blood cells, but instead has hemolymph that flows in an ‘open circulatory system’ – this hemolymph contains KLH as its major glycoprotein. KLH carries oxygen to all cells and tissues that are bathed in the hemolymph. The protein is a metalloprotein with chelated copper (2 per subunit), and when bound to oxygen, KLH turns blue. Each subunit of KLH has ~3,400 amino acids, with a large amount of carbohydrate linked to the amino acids; the carbohydrate content amounts to ~3-4% (w/w). KLH has an apparent size of ~390 KDa and its subunit dimers are ~600-800 kDa; however, the common aggregates of KLH subunits are truly gigantic, and have sizes of 4,500 kDa-13,000 kDa and exist in a barrel-shape with multiple oligomeric structures (1).
Because of its unique peptide sequence and unusual glycosylation, KLH is remarkably immunogenic in humans and animals; it is often used as a ’carrier’ with haptens to generate T-cell dependent antibodies (2). It was first given to human in immunization studies in 1967, as a potent antigen, which was used to explore potential immunosuppressive therapies (3). Since then, many studies (4) have been done using KLH as a non-specific immunostimulant and adjuvant, and it is gaining interest in regard to cancer vaccines (5).
The glycans on KLH are incredibly heterogeneous and immunogenic. KLH glycans lack sialic acid, but contain unusual N-glycans (of which there are ~8 per subunit) with high amounts of fucose, xylose, N-acetylgalactosamine, N-acetylglucosamine, galactose, and mannose, and many unusual linkages of sugars, such as Galβ1–6Man, Fucα1-3GalNAc, and Fucα1-2Fuc, and Galβ1-4Fuc (6-9). By cryo-electron microscopy, KLH appears to be a gigantic complex in which the glycans are observed as 6 protrusions per subunit, indicating a total of “120 sugar trees”, as the authors termed them, in KLH1 (1). Interestingly, many of the carbohydrate antigens, such as the difucosylated LacdiNAc epitope - Fuc(α1-3)GalNAc(β1-4)[Fuc(α1-3)]GlcNAcβ1-R - cross-react with glycoconjugates from the trematode parasite Schistosoma mansoni (6,10).
References
- Gatsogiannis, C., and Markl, J. (2009) Keyhole limpet hemocyanin: 9-A CryoEM structure and molecular model of the KLH1 didecamer reveal the interfaces and intricate topology of the 160 functional units. J Mol Biol 385, 963-983
- Swaminathan, A., Lucas, R. M., Dear, K., and McMichael, A. J. (2014) Keyhole limpet haemocyanin - a model antigen for human immunotoxicological studies. Br J Clin Pharmacol 78, 1135-1142
- Swanson, M. A., and Schwartz, R. S. (1967) Immunosuppressive therapy. The relation between clinical response and immunologic competence. N Engl J Med 277, 163-170
- Curtis, J. E., Hersh, E. M., Harris, J. E., McBride, C., and Freireich, E. J. (1970) The human primary immune response to keyhole limpet haemocyanin: interrelationships of delayed hypersensitivity, antibody response and in vitro blast transformation. Clin Exp Immunol 6, 473-491
- Harris, J. R., and Markl, J. (1999) Keyhole limpet hemocyanin (KLH): a biomedical review. Micron 30, 597-623
- Geyer, H., Wuhrer, M., Resemann, A., and Geyer, R. (2005) Identification and characterization of keyhole limpet hemocyanin N-glycans mediating cross-reactivity with Schistosoma mansoni. J Biol Chem 280, 40731-40748
- Wuhrer, M., Robijn, M. L., Koeleman, C. A., Balog, C. I., Geyer, R., Deelder, A. M., and Hokke, C. H. (2004) A novel Gal(beta1-4)Gal(beta1-4)Fuc(alpha1-6)-core modification attached to the proximal N-acetylglucosamine of keyhole limpet haemocyanin (KLH) N-glycans. Biochem J 378, 625-632
- Kurokawa, T., Wuhrer, M., Lochnit, G., Geyer, H., Markl, J., and Geyer, R. (2002) Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(beta1-6)Man-motifs. Eur J Biochem 269, 5459-5473
- Staudacher, E. (2021) Mollusc N-glycosylation: Structures, Functions and Perspectives. Biomolecules 11
- Markl, J., Nour el Din, M., Winter-Simanowski, S., and Simanowski, U. A. (1991) Specific IgG activity of sera from Egyptian schistosomiasis patients to keyhole limpet hemocyanin (KLH). Naturwissenschaften 78, 30-31