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Because of the composition and structural complexity of crustacean shells, their color change mechanism during thermal processing remains unclear. This study identified and characterized two intrinsic protein components, hemocyanin (Lv-Hc) and β-1,3-glucan-binding protein (Lv-BGBP) from Litopenaeus vannamei shrimp shells by a combination of ion-exchange chromatography, gel filtration, and mass spectrometry. It was found that a mixture of Lv-Hc, a gray protein, and Lv-BGBP (which is a natural astaxanthin-binding protein with a red color) is responsible for the brown color of fresh shrimp shells. Upon heating to 100 °C, the mixture of these proteins turned red, mimicking the color change observed in cooked shrimp shells. This transition is attributed to the extremely high thermal stability of Lv-BGBP, which has the ability to protect astaxanthin from thermal induced degradation. These findings provide significant insights into the molecular mechanism governing shrimp shell coloration, advancing our understanding of crustacean biochemistry.

To identify the key amino acids (AAs) affecting the allergenicity of hemocyanin (HC) allergens from Chinese mitten crabs, in this study, two epitopes, P1-SHFTGSKSNPEQR and P2-LSPGANTITR were employed and four potential key AAs (P1: F3 and N9 and P2: N6 and R10) were predicted. Mast cell and mouse models revealed that four mutants induced lower levels of immunoglobulin E (IgE) and Th2 type cytokines (15.47-49.89 %), proving that F3, N9, N6, and R10 were the key AAs of two epitopes. Mutants reduce allergic responses via the Th2 pathway. However, the roles of every key AA affecting allergenicity were different (P1-F3 > N9 and P2-N6 > R10). In addition, lower transport and higher efflux were observed in the mutants during transport absorption by Caco-2 cells. The allergenicity of HC was stronger when the transport absorption efficiency of epitopes and mutants was higher and their efflux was lower. Our study provides a novel method for revealing the allergenic molecular mechanisms of food allergens., Competing Interests: Declaration of competing interest The authors attested that there were no conflicts of interest concerning this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten. Three carrier proteins e.g. KLH (Keyhole Limpet hemocyanin), BSA (bovine serum albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the cost of obtaining Keyhole Limpet is high and the solubility of KLH is not as well as the other carriers, especially after hapten conjugation. Here, we extracted the shrimp hemocyanin (SHC) from Litopenaeus vannamei (L. vannamei), which is a commonly sea product worldwide. The high pure SHC could be acquired by two-step purification, with a production yield of > 1 g proteins (98% pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Meanwhile, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Furthermore, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and the subsequent antibodies were confirmed to be used in western blot, immunofluorescence and immunohistochemistry. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development., (© 2024. The Author(s).)

Mollusk hemocyanins, among the largest known proteins, are used as immunostimulants in biomedical and clinical applications. The hemocyanin of the Chilean gastropod Concholepas concholepas (CCH) exhibits unique properties, which makes it safe and effective for human immunotherapy, as observed in animal models of bladder cancer and melanoma, and dendritical cell vaccine trials. Despite its potential, the structure and amino acid sequence of CCH remain unknown. This study reports two sequence fragments of CCH, representing three complete functional units (FUs). We also determined the high-resolution (1.5 Å) X-ray crystal structure of an "FU-g type" from the CCHB subunit. This structure enables in-depth analysis of chemical interactions at the copper-binding center and unveils an unusual, truncated N-glycosylation pattern. These features are linked to eliciting more robust immunological responses in animals, offering insights into CCH's enhanced immunostimulatory properties and opening new avenues for its potential applications in biomedical research and therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hemocyanin, an oxygen-transport protein, is widely distributed in the hemolymph of marine arthropods and mollusks, playing an important role in their physiological processes. Recently, hemocyanin has been recognized as a multifunctional glycoprotein involved in the immunological responses of aquatic invertebrates. Consequently, the link between hemocyanin functions and their potential applications has garnered increased attention. This review offers an integrated overview of hemocyanin's structure, physicochemical characteristics, and bioactivities to further promote the utilization of hemocyanin derived from marine products. Specifically, we review its implication in two aspects of food and aquaculture industries: quality and health. Hemocyanin's inducible phenoloxidase activity is thought to be an inducer of melanosis in crustaceans. New anti-melanosis agents targeted to hemocyanin need to be explored. The red-color change observed in shrimp shells is related to hemocyanin, affecting consumer preferences. Hemocyanin's adaptive modification in response to the aquatic environment is available as a biomarker. Additionally, hemocyanin is endowed with bioactivities encompassing anti-microbial, antiviral, and therapeutic activities. Hemocyanin is also a novel allergen and its allergenic features remain incompletely characterized., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models., Materials and Methods: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed., Results: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression., Discussion: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and β-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-β-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.