Keyhole limpet hemocyanin is an extremely large, heterogeneous glycosylated protein consisting of subunits with a molecular weight of 350,000 and 390,000 in aggregates with molecular weights of 4,500,000-13,000,000. That, in vivo, increases antigenic immune responses to haptens and other weak antigens such as idiotype proteins. Each domain of aKLH subunit contains two copper atoms that together bind a single oxygen molecule (O2). When oxygen is bound to hemocyanin, the molecule takes on a distinctive transparent, opalescent blue color. The KLH protein is potently immunogenic yet safe in humans and is therefore highly prized as a vaccine carrier protein. The large and highly glycosylated KLH protein cannot be reproduced synthetically. It is available only as a purified biological product from the Keyhole Limpet Megathura crenulata.
IMMUNOTHERAPY AND BLADDER CANCER TREATMENT
Chemo- and immunotherapeutic approaches have been used to prevent recurrence of transitional cell carcinoma (TCC), the most common type of superficial bladder cancer (SBC). The bacillus Calmette-GuÃ©rin (BCG) vaccine for tuberculosis, which consists of an attenuated form of Mycobacterium bovis, is the most commonly used immunotherapeutic agent. Despite the successful results achieved with BCG, its serious side effects have led researchers to investigate other immunostimulatory substances.
In the early 1970s, Olsson and collaborators reported that subcutaneous stimulation with keyhole limpet hemocyanin (KLH) from the Californian marine gastropod Megathura crenulata significantly reduced SBC recurrence frequency in TCC patients without any toxic side effects, making it ideal for long-term repetitive treatments. These results provided promising support for the use of mollusk hemocyanins as alternative agents in SBC immunotherapy. Hemocyanins, blue respiratory glycoproteins that were discovered in 1878 by L©on Fredericq, are found freely dissolved in the blood of some mollusks and arthropods. These proteins are giant structures with molecular weights between 4 and 8 MDa, and they exhibit some of the most complex and sophisticated quaternary structures known.
Hemocyanins are part of the type-3 group of copper proteins that includes phenoloxidases and tyrosinases). These proteins contain active copper-containing sites in which the Cu(I,I) state is oxidized to the Cu(II,II) state, thus accounting for their distinctive deep blue color. Because of these roperties, the biochemistry of hemocyanins has been intensively studied. The pioneering work of Weigle in the 1960s on the immunochemical properties of KLH demonstrated its remarkable immunostimulatory properties in an experimental animal model. These results were quickly incorporated into clinical studies to evaluate its immunological effects.
USE IN BIOTECHNOLOGY
Keyhole limpet hemocyanin (KLH) is used extensively as a carrier protein in the production of antibodies for research, biotechnology andtherapeutic applications. Haptens are substances with a low molecular weight such as peptides, small proteins and drug molecules that are generally not immunogenic and require the aid of a carrier protein to stimulate a response from the immune system in the form of antibody production. KLH is the most widely employed carrier proteins for this purpose.
KLH is an effective carrier protein for several reasons. Its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues for coupling haptens, allows a high hapten:carrier protein ratio increasing the likelihood of generating hapten-specific antibodies. In addition, because KLH is derived from the limpet, a gastropod, it is phylogenetically distant from mammalian proteins, thus reducing false positives in immunologically based research techniques in mammalian model organisms.
KLH may also be a challenging molecule to work with because of its propensity to aggregate and precipitate. Aggregates remain immunogenic, but limit the ability to conjugate haptens and are difficult to manipulate in the laboratory. A high quality KLH preparation with the clear opalescent blue color is the best indicator of KLH solubility.
Sources: enature.com, Bill Bushing, Decker & Tuczek, 2000, van Holde & Miller, 1995