Ca2+-dependent Antifreeze Proteins

Abstract

The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth. was bound to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca 2؉ and, consequently, adopts a conformation that is essential for its ice-binding activity.Many marine teleost fishes are protected from freezing in icy sea water by antifreeze proteins (AFPs) 1 or glycoproteins (AFGPs). These proteins lower the freezing points of solutions in a noncolligative manner by inhibiting the growth of ice crystals (Ananthanarayanan, 1989;Davies and Hew, 1990). Four distinct types of antifreezes have been found in fishes, and each has a narrow phyletic distribution. The AFGPs are found in the cods and in the nototheniids (see Davies and Hew (1990) and references therein), and they consist of a series of repeated tripeptide units (Ala-Ala-Thr) with an O-linked disaccharide linked to each Thr residue. The type I AFPs, found in sculpins and in righteye flounders, are Ala-rich amphipathic ␣-helices (Davies and Hew, 1990). The type II AFPs are larger proteins with a folded structure and are found in smelt (Osmerus mordax), herring (Clupea harengus harengus), and sea raven (Hemitripterus americanus) (Ewart et al., 1992;Ng and Hew, 1992;Ewart and Fletcher, 1993). Type III AFP is a protein with a ␤ sandwich structure and appears limited to eel pouts (Sön-nichsen et al., 1993).The type II AFPs from sea raven, smelt, and herring share a high protein sequence identity. All three proteins are homologous to the carbohydrate recognition domains (CRDs) of Ca 2ϩ -dependent (C-type) lectins and correspond to the group VII C-type lectin family (Drickamer and Taylor, 1993). Multiple sequence alignment of the sea raven AFP with C-type CRDs and modeling of the AFP using coordinates from the CRD of rat mannose-binding protein (Weis et al., 1991a) has confirmed the structural similarity of the type II AFP to the C-type CRD fold (Sönnichsen et al., 1995). However, in contrast to the lectins, the AFPs do not bind to carbohydrates .Two Ca 2ϩ -binding sites were identified in the crystal structure of a C-type CRD from rat mannose-binding protein (MBP), and a single site was identified in the CRD of E-selectin (Weis et al., 1991a;Graves et al., 1994). The Ca 2ϩ -binding site that is shared by the selectin and the MBP was shown to be the carbohydrate-binding surface in a crystal structure of an oligosaccharide-MBP complex (Weis et al., 1992). Like the C-type lectins, the herring and smelt AFPs require Ca 2ϩ for their activity (Ewart et al., 1992;Ewart and Fletcher, 1993). In order to understand the role of Ca 2ϩ in the structural integrity and activity of these AFPs and the structural alterations underlying the evolution from carbohydrate to ice binding, we examined the role of Ca 2ϩ in the conformation and the antifreeze activity of the herring AFP (hAFP). Our findings show that hAFP contains a single Ca . EXPERIMENTAL PROCEDURESMaterial...

Interaction of ice binding proteins with ice, water and ions

“…51 Interestingly, the TH activity of the protein is significantly lower in the presence of other divalent ions, which also leads to different ice crystal shapes. 52 Globular type III fish AFPs are devoid of cysteine. They are subdivided in quaternary aminoethyl (QAE) and sulfopropyl (SP) isoforms, based on sequence similarity and isoelectric point: QAE isoforms adhere to QAE sephadex ion exchange resin, while SP isoforms adhere to SP sephadex ion exchange resins.…”

Mechanism of Ca2- and Monosaccharide Binding to a C-type Carbohydrate-recognition Domain of the Macrophage Mannose Receptor

“…Either the MBP-A pattern of ligation or the alternative ligation pattern seen in E-selectin could be utilized. However, the presence of the ligands for the conserved Ca 2ϩ in a CRD , probably at this site, but it does not bind carbohydrate (25). Equally, some of the more divergent groups of C-type lectins, including the type II transmembrane proteins found on natural killer cells, may utilize a completely different mechanism for binding sugar and/or Ca 2ϩ , since these proteins do not contain the ligands for the conserved Ca 2ϩ (4,26,27 , are predicted, based on the mutagenesis results, to be involved in ligation of the auxiliary Ca 2ϩ (Fig.…”

Calcium Interacts with Antifreeze Proteins and Chitinase from Cold-Acclimated Winter Rye

“…, a type II AFP adopts a new conformation that creates the icebinding domain (Ewart et al, 1996(Ewart et al, , 1998(Ewart et al, , 1999(Ewart et al, , 2000Achenbach and Ewart, 2002). However, Ca 21 had no effect on antifreeze activity in unfrozen winter rye extracts (Fig.…”