Molecular basis for polysialylation: A novel polybasic polysialyltransferase domain (PSTD) of 32 amino acids unique to the α2,8-polysialyltransferases is essential for polysialylation

Abstract

To determine the molecular basis of eukaryotic polysialylation, the function of a structurally unique polybasic motif of 32 amino acids (pI approximately 12) in the polysialyltransferases (polySTs), ST8Sia II (STX and ST8Sia IV (PST) was investigated. This motif, designated the "polysialyltransferase domain" (PSTD), is immediately upstream of the sialylmotif S (SM-S). PolyST activity was lost in COS-1 mutants in which the entire PSTD in ST8Sia IV was deleted, or in mutants in which 10 and 15 amino acids in either the N- or C- terminus of PSTD were deleted. Site-directed mutagenesis showed that Ile(275), Lys(276) and Arg(277) in the C-terminus of PSTD in ST8Sia IV, which is contiguous with the N-terminus of sialylmotif-S, were essential for polysialylation. Arg(252) in the N-terminus segment of the PSTD was also required, as was the overall positive charge. Thus, multiple domains in the polySTs can influence their activity. Immunofluorescent microscopy showed that the mutated proteins were folded correctly, based on their Golgi localization. The structural distinctness of the conserved PSTD in the polySTs, and its absence in the mono- oligoSTs, suggests that it is a "polymerization domain" that distinguishes a polyST from a monosialyltransferases. We postulate that the electrostatic interaction between the polybasic PSTD and the polyanionic polySia chains may function to tether nascent polySia chains to the enzyme, thus facilitating the processive addition of new Sia residues to the non-reducing end of the growing chain. In accord with this hypothesis, the polyanion heparin was shown to inhibit recombinant human ST8Sia II and ST8Sia IV at 10 microM.

Sequences Prior to Conserved Catalytic Motifs of Polysialyltransferase ST8Sia IV Are Required for Substrate Recognition

“…Troy and colleagues (60) characterized a stretch enriched in basic residues termed the polysialyltransferase domain (PSTD) (residues 246 -277 in PST and 261-292 in STX), and found that the overall positive charge of this region was required for NCAM polysialylation. They postulated that the PSTD facilitates processivity of the polysialylation process by interacting with the growing, negatively charged polySia chain (60). However, our analysis of these critical PSTD residues suggested that this region is important for polyST catalytic activity (61).…”

“…The PSTD is a stretch of 32 amino acids (residues 246 -277 in PST) that is contiguous with the SMS and is conserved in the two polySTs (60). Previous work demonstrated that selected residues in the PSTD are required for NCAM polysialylation (60,61).…”

Molecular evolution and expression of zebrafish St8SiaIII, an alpha‐2,8‐sialyltransferase involved in myotome development

“…1C). All St8SiaIII proteins, however, lack 2 of 4 amino acids required for polysialylation of target proteins as determined by Nakata et al (2006) (exchanged: R-252 and I-275; conserved: K-276 and R-277; positions refer to human St8SiaIV).…”

“…A high degree of conservation within the linkage specificity domain (amino acid residues 198 -207) indicates that St8SiaSiaIII, such as St8SiaII and St8SiaIV, catalyzes the formation of ␣-2,8-linked sugars and thus is a St8-sialyltransferase Patel and Balaji, 2006). The fact that the PSTD domain of St8SiaIII in zebrafish and other species lacks 2 of 4 amino acids required for polysialylation of target proteins (Nakata et al, 2006) supports in vitro biochemical data, which point towards a function of vertebrate St8SiaIII enzymes as monoor oligosialyltransferase rather that as polysialyltransferases (Angata et al, 2000;Sato et al, 2002). This idea is corroborated by the fact that the major expression domain of St8SiaIII in zebrafish is devoid of polysialic acid.…”

“…It is formed by four conserved sialyltransferase motifs (SMs): SM-L (large) is mainly involved in recognition of the sugar donor CMP-Neu5Ac; SM-S (small) is involved in donor and acceptor binding; SM-III (Jeanneau et al, 2004) and SM-VS (very small) play important roles in the catalytic activity, which have not yet been unraveled in detail. A polysialyltransferase domain (PSTD), located immediately upstream of SM-L in polysialyltransferases, is obligatorily required for polysialylation of target molecules (Nakata et al, 2006). The catalytic activity of STs depends on the formation of two disulfide bonds, through which these motifs are arranged in close proximity of each other (Angata et al, 2001; reviewed in Harduin- Lepers et al, 2005;Patel andBalaji, 2006, Nakata et al, 2006).…”