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

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Abstract

To determine the molecular basis of eukaryotic polysialylation, the function of a structurally unique polybasic motif of 32 amino acids (pI∼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 Ile275, Lys276 and Arg277 in the C-terminus of PSTD in ST8Sia IV, which is contiguous with the N-terminus of sialylmotif-S, were essential for polysialylation. Arg252 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 μM.

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Abbreviations

Sia:

sialic acid

polySia:

polysialic acids

N-CAM:

neural cell adhesion molecules

DP:

degree of polymerization

ST8Sia II (STX):

ST8 α-N-acetylneuraminide α-2,8-sialyltransferase II

ST8Sia IV (PST):

ST8 α-N-acetylneuraminide α-2,8-sialyltransferase IV

PolySTs:

polysialyltransferases

Endo-N:

Endo-N-acylneuraminidase

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  1. Department of Biochemistry and Molecular Medicine, University of California School of Medicine, Davis, CA, 95616, USA

    Daisuke Nakata, Lirong Zhang & Frederic A. Troy II

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  1. Daisuke Nakata
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  2. Lirong Zhang
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  3. Frederic A. Troy II
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Correspondence to Frederic A. Troy II.

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Nakata, D., Zhang, L. & Troy, F.A. Molecular basis for polysialylation: A novel polybasic polysialyltransferase domain (PSTD) of 32 amino acids unique to the α2,8-polysialyltransferases is essential for polysialylation. Glycoconj J 23, 423–436 (2006). https://doi.org/10.1007/s10719-006-6356-5

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  • DOI: https://doi.org/10.1007/s10719-006-6356-5

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