In Vitro Studies of Antifreeze Glycoprotein (AFGP) and a C-Linked AFGP Analogue

Abstract

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in deep sea Teleost fish. These compounds have the ability to depress the freezing point of the organism such that it can survive the subzero temperatures encountered in its environment. This physical property is very attractive for the cryopreservation of cells, tissues, and organs. Recently, our laboratory has designed and synthesized a functional carbon-linked (Clinked) AFGP analogue (1) that demonstrates tremendous promise as a novel cryoprotectant. Herein we describe the in Vitro effects and interactions of C-linked AFGP analogue 1 and native AFGP 8. Our studies reveal that AFGP 8 is cytotoxic to human embryonic liver and human embryonic kidney cells at concentrations higher than 2 and 0.63 mg/mL, respectively, whereas lower concentrations are not toxic. The mechanism of this cytotoxicity is consistent with apoptosis because caspase-3/7 levels are significantly elevated in cell cultures treated with AFGP 8. In contrast, C-linked AFGP analogue 1 displayed no in Vitro cytotoxicity even at high concentrations, and notably, caspase-3/7 activities were suppressed well below background levels in cell lines treated with 1. Although the results from these studies limit the human applications of native AFGP, they illustrate the benefits of developing functional C-linked AFGP analogues for various medical, commercial and industrial applications.

Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing

“…Bovine sperm and mouse oocytes 19 appear to benefit from AFP during cryopreservation but AFPs reduce the viability of mouse sperm under similar conditions 16 . There are also indications that AF(G)Ps are cytotoxic to mammalian cells, plus the use of non-host proteins may create immunogenicity issues 20 . Upon analysis of the cryopreservation process, it has been determined that one of the major causes of cell death is actually ice recrystallization (growth) during the thawing process, rather than cell death during freezing 21,22 .…”

Design and Synthesis of Antifreeze Glycoproteins and Mimics

“…Furthermore, analogue 7 showed little or no in vitro cytotoxicity, even at high concentrations, and was able to penetrate cells and inhibit apoptotic death. [80] This result contrasts with AFGP8, which is cytotoxic to human embryonic liver and kidney cells, a property that is likely to be related to a rapid internalisation by human liver cells through ATP-dependent endocytosis. Although the Clinked analogue is also rapidly internalised by cells, the lack of cytotoxicity is an important advantage for applications like cryoprotection and hypothermal storage, and might also be effective in preventing cold-induced apoptosis.…”

“…Although the Clinked analogue is also rapidly internalised by cells, the lack of cytotoxicity is an important advantage for applications like cryoprotection and hypothermal storage, and might also be effective in preventing cold-induced apoptosis. [80] Many compounds, including the structurally simple polymer polyethenol, exhibit RI activity. In light of the RI activity exhibited by the C-linked AFGP mimics and the clear dependence of RI activity on the distance between the carbohydrate and the peptide backbone, Gibson et al [85] studied a structurally diverse series of well-characterised water-soluble polymers in order to determine the structural features required for recrystallisation inhibition.…”

C-Linked Antifreeze Glycoprotein (C-AFGP) Analogues as Novel Cryoprotectants

“…1 H NMR (CDCl 3 , 400 MHz) δ 4.41 (brs, 1H), 4.32 (m, 1H), 4.25 (dd, J = 9.7, 5.4 Hz, 1H), 4.06 (dd, J = 12.9, 2.0 Hz, 1H), 3.85 (d, J = 12.9 Hz, 1H), 3.67 (dd, J = 9.9, 2.9 Hz, 1H), 3.59 (dd, J = 10.7, 3.3 Hz, 1H), 3. 23 ((S)-3-(N-(9-fluorenylmethyloxycarbonyl)amino)-4-(1 0 -deoxy-2 0 ,3 0 ;4 0 ,6 0 -di-O-isopropylidene-R-D-galactopyranosyl)-butanol (18). To a solution of 17 (1.30 g, 3.9 mmol) and NaHCO 3 (0.65 g, 7.8 mmol, 2 equiv.)…”