Keyhole limpet hemocyanin: Structural and functional characterization of two different subunits and multimers

Swerdlow, Richard D.; Ebert, Ray F.; Lee, Psyche; Bonaventura, Celia; Miller, Karen I.

doi:10.1016/0305-0491(95)02091-8

Cited by 74 publications

(60 citation statements)

References 25 publications

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“…The percentage entrapment of BSA observed is similar to that reported in previous studies investigating the encapsulation of human serum albumin by human carrier erythrocytes [28,29]. KLH consists of two different polypeptides of molecular weights 449,000 and 392,000 Da, existing in aggregation states ranging between 4.5 x 10 5 and 1.3 x 10 7 Da depending on pH and divalent ion concentration [30,31]. Based on the observations of Deloach et al [32] who reported an entrapment efficiency of 39% for β-galactosidase (molecular weight 595,000 Da), an entrapment efficiency of 35% would suggest that KLH existed in a low state of aggregation during the hypo-osmotic dialysis procedure.…”

Section: Discussionsupporting

confidence: 87%

The mouse immune response to carrier erythrocyte entrapped antigens

Murray

Pearson

Fairbanks

et al. 2006

Vaccine

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Section: Discussionsupporting

confidence: 87%

The mouse immune response to carrier erythrocyte entrapped antigens

Murray

Pearson

Fairbanks

et al. 2006

Vaccine

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“…The decamers in gastropod hemocyanins may associate in pairs to form colossal molecules called didecamers, with molecular masses of ∼8 MDa (23). The hemocyanins used in this study present clear structural differences at this level, and experimental data have demonstrated that the KLH preparation is made up of two independent isoforms that coexist in variable proportions in the hemolymph of the animals, each composed of one type of subunit (24). In contrast, the two subunits of CCH are intermingled in the molecule, forming heterodidecamers (7).…”

mentioning

confidence: 91%

Hemocyanins Stimulate Innate Immunity by Inducing Different Temporal Patterns of Proinflammatory Cytokine Expression in Macrophages

Zhong

Arancibia

Born

et al. 2016

The Journal of Immunology

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Hemocyanins induce a potent Th1-dominant immune response with beneficial clinical outcomes when used as a carrier/adjuvant in vaccines and nonspecific immunostimulant in cancer. However, the mechanisms by which hemocyanins trigger innate immune responses, leading to beneficial adaptive immune responses, are unknown. This response is triggered by a proinflammatory signal from various components, of which macrophages are an essential part. To understand how these proteins influence macrophage response, we investigated the effects of mollusks hemocyanins with varying structural and immunological properties, including hemocyanins from Concholepas concholepas, Fissurella latimarginata, and Megathura crenulata (keyhole limpet hemocyanin), on cultures of peritoneal macrophages. Hemocyanins were phagocytosed and slowly processed. Analysis of this process showed differential gene expression along with protein levels of proinflammatory markers, including IL-1β, IL-6, IL-12p40, and TNF-α. An extended expression analysis of 84 cytokines during a 24-h period showed a robust proinflammatory response for F. latimarginata hemocyanin in comparison with keyhole limpet hemocyanin and C. concholepas hemocyanin, which was characterized by an increase in the transcript levels of M1 cytokines involved in leukocyte recruitment. These cytokine genes included chemokines (Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3), ILs (Il1b and Ifng), growth factors (Csf2 and Csf3), and TNF family members (Cd40lg). The protein levels of certain cytokines were increased. However, every hemocyanin maintains downregulated key M2 cytokine genes, including Il4 and Il5. Collectively, our data demonstrate that hemocyanins are able to trigger the release of proinflammatory factors with different patterns of cytokine expression, suggesting differential signaling pathways and transcriptional network mechanisms that lead to the activation of M1-polarized macrophages.

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“…The demonstrated existence of different conformational states of KLH was expected from the fact that KLH with its 160 oxygen binding centers binds dioxygen, cooperatively serving as an oxygen carrier in vivo (14). The radii of gyration were determined from the SAXS data to be 163.8 Ϯ 0.3 and 164.7 Ϯ 0.3 Å for oxygenated and deoxygenated KLH1, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…KLH has widely been used as an immunostimulant, hapten carrier, and valuable agent in the treatment of cancer. However, not much is known regarding the immunological (6 -10) and biochemical as well as structural properties (11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 99%

Small-angle X-ray Scattering-based Three-dimensional Reconstruction of the Immunogen KLH1 Reveals Different Oxygen-dependent Conformations

Hartmann

Bongers

Decker

2004

Journal of Biological Chemistry

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For decades the respiratory protein keyhole limpet hemocyanin (KLH1) from the marine gastropod Megathura crenulata has been used widely as a potent immunostimulant, useful hapten carrier, and valuable agent in the treatment of bladder carcinoma. Although much information on the immunological properties of KLH1 is available, biochemical and structural data are still incomplete. Small-angle x-ray scattering revealed the existence of two conformations, an oxy state being slightly more compact than the deoxy state. Based on small-angle scattering curves, a newly developed Monte Carlo algorithm delivered a surface representation of proteins. The massive changes of the surfaces of reconstructed didecameric KLH1 molecules are explained as a twist of the two non-covalently associated decameric half-molecules. Upon oxygenation, the KLH1 molecule becomes longer and skinnier. This study provides the first real evidence how a molluscan hemocyanin changes conformation during an allosteric transition.

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Keyhole limpet hemocyanin: Structural and functional characterization of two different subunits and multimers

Cited by 74 publications

References 25 publications

The mouse immune response to carrier erythrocyte entrapped antigens

The mouse immune response to carrier erythrocyte entrapped antigens

Hemocyanins Stimulate Innate Immunity by Inducing Different Temporal Patterns of Proinflammatory Cytokine Expression in Macrophages

Small-angle X-ray Scattering-based Three-dimensional Reconstruction of the Immunogen KLH1 Reveals Different Oxygen-dependent Conformations

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