Sains Malaysiana 54(5)(2025): 1269-1280

http://doi.org/10.17576/jsm-2025-5405-06

Interplay between Collagen Hydrolysates and the Ability of FKBP35 from Plasmodium knowlesi in Preventing Insulin Aggregation

 (Interaksi antara Kolagen Hidrolisat dan Keupayaan FKBP35 daripada Plasmodium knowlesi dalam Mencegah Pengagregatan Insulin)

NUR ILIYANA ILLANG¹, CARLMOND GOH KAH WUN¹, MUHAMMAD ARIFIN² & CAHYO BUDIMAN^1,2,*

¹Biotechnology Research Institute, Universiti Malaysia Sabah, Jln UMS, 88400 Kota Kinabalu, Sabah, Malaysia

 ²Department of Animal Production and Technology, Faculty of Animal Sciences, IPB University, Kampus IPB Darmaga IPB Darmaga Bogort 16680, Indonesia

Received: 17 November 2023/Accepted: 10 February 2025

Abstract

FK506-binding protein 35 (FKBP35) from Plasmodium knowlesi (Pk-FKBP35) is a potential target for combating the surge in simian malaria cases. While previously speculated to hinder protein synthesis aggregation within parasite cells, this study seeks to experimentally validate the capacity of Pk-FKBP35 to avert protein aggregation. Additionally, it aims to examine the influence of collagen hydrolysates (CH) on this ability. Initially, Pk-FKBP35 was overexpressed in Escherichia coli BL21(DE3) and subsequently purified. To assess its capacity for preventing aggregation, a dithiothreitol (DTT)-induced insulin aggregation assay was conducted and observed via SDS-PAGE. The findings showed a concentration-dependent inhibition by Pk-FKBP35 against DTT-induced insulin aggregation. At the concentration of 0.75 mg/mL of Pk-FKBP35, the amount of soluble insulin was increased to about 5-fold higher. Interestingly, in the presence of FK506, Pk-FKBP35's ability to prevent insulin aggregation remains intact. Since FK506 is known to specifically bind to the catalytic domain of Pk-FKBP35, this suggests that the region responsible for the protein's aggregation prevention activity is independent from the catalytic domain.  Moreover, when coupled with CH derived from bovine, bone broth, fish, and swine, Pk-FKBP35's effectiveness in preventing DTT-induced insulin aggregation was attenuated, albeit to varying degrees. Notably, swine and bone broth CH exhibited superior inhibition of aggregation prevention compared to bovine and fish CH. This study validates Pk-FKBP35's capability to impede protein aggregation, showcasing a promising potential for inhibition by CH, particularly those sourced from swine and bone broth.

Keywords: Collagen hydrolysate; FKBP35; peptidyl-prolyl cis-trans isomerase (PPIase); Plasmodium knowlesi; zoonotic malaria

Abstrak

Protein pengikat-FK506 daripada Plasmodium knowlesi (Pk-FKBP35) dipercayai berpotensi untuk dibangunkan sebagai ubat anti-malaria bagi melawan kes zoonotik malaria yang semakin membimbangkan. Walaupun sebelum ini dikatakan dapat menghalang penggumpalan semasa sintesis protein dalam sel parasit, kajian ini bertujuan untuk mengesahkan secara uji kaji keupayaan Pk-FKBP35 untuk mencegah penggumpalan protein. Selain itu, kajian ini juga bertujuan untuk meneliti pengaruh hidrolisat kolagen (CH) terhadap keupayaan tersebut. Dengan itu, Pk-FKBP35 diekspresikan secara berlebihan di dalam E. coli (BL21) dan ditulenkan. Keupayaan menghalang penggumpalan protein diperhatikan menggunakan insulin sebagai model substrat yang dinyahaslikan menggunakan ditiotreitol (DTT) dan dipantau secara visual melalui elektroforesis gel natrium dodesil sulfat-poliakrilamida (SDS-PAGE). Keputusan menunjukkan bahawa Pk-FKBP35 berupaya menghalang penggumpalan insulin secara kebergantungan kepekatan. Bilangan insulin yang larut meningkat lima kali ganda dengan kehadiran 0.75 mg/mL Pk-FKBP35. Menariknya, kehadiran FK506 tidak menjejaskan kemampuan Pk-FKBP35 untuk mencegahpenggumpalan insulin. Memandangkan FK506 diketahui melekat secara khas pada domain katalitik Pk-FKBP35, ini menunjukkan bahawa kawasan yang bertanggungjawab untuk aktiviti pencegahanpenggumpalan protein adalah berasingan daripada domain katalitik tersebut. Dalam masa yang sama, kehadiran CH daripada lembu, tulang, ikan dan khinzir berupaya mengurangkan pencegahan penggumpalan insulin yang dinyahaslikan menggunakan DTT dengan kadar yang berbeza. CH khinzir dan tulang mencegah keupayaan Pk-FKBP35 lebih baik berbanding CH daging dan ikan. Kajian ini membuktikan Pk-FKBP35 mempunyai keupayaan mencegah penggumpalan protein dan keupayaan ini boleh direncatkan dengan kehadiran CH.

Kata kunci: FKBP35; hidrolisat kolagen; isomerase cis-trans peptidil-prolil (PPIase); Plasmodium knowlesi; zoonotik malaria

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*Corresponding author; email: cahyo@ums.edu.my