Sequences prior to conserved catalytic motifs of polysialyltransferase ST8Sia IV are required for substrate recognition

Abstract

Polysialic acid on the neural cell adhesion molecule (NCAM) modulates cell-cell adhesion and signaling, is required for proper brain development, and plays roles in neuronal regeneration and the growth and invasiveness of tumor cells. Evidence indicates that NCAM polysialylation is highly protein-specific, requiring an initial polysialyltransferase-NCAM protein-protein interaction. Previous work suggested that a polybasic region located prior to the conserved polysialyltransferase catalytic motifs may be involved in NCAM recognition, but not overall enzyme activity (Foley, D. A., Swartzentruber, K. G., and Colley, K. J. (2009) J. Biol. Chem. 284, 15505-15516). Here, we employ a competition assay to evaluate the role of this region in substrate recognition. We find that truncated, catalytically inactive ST8SiaIV/PST proteins that include the polybasic region, but not those that lack this region, compete with endogenous ST8SiaIV/PST and reduce NCAM polysialylation in SW2 small cell lung carcinoma cells. Replacing two polybasic region residues, Arg(82) and Arg(93), eliminates the ability of a full-length, catalytically inactive enzyme (PST H331K) to compete with SW2 cell ST8SiaIV/PST and block NCAM polysialylation. Replacing these residues singly or together in ST8SiaIV/PST substantially reduces or eliminates NCAM polysialylation, respectively. In contrast, replacing Arg(82), but not Arg(93), substantially reduces the ability of ST8SiaIV/PST to polysialylate neuropilin-2 and SynCAM 1, suggesting that Arg(82) plays a general role in substrate recognition, whereas Arg(93) specifically functions in NCAM recognition. Taken together, our results indicate that the ST8SiaIV/PST polybasic region plays a critical role in substrate recognition and suggest that different combinations of basic residues may mediate the recognition of distinct substrates.

FIGURE 1.

Schematic of ST8Sia IV/PST and PST mutants. Shown are full-length ST8SiaIV/PST and the truncated and mutant proteins used in this study. All truncated PST mutants lack the sialyl motifs (SML, SMS, motif III, SMVS) found in full-length PST, and hence are catalytically inactive. Soluble PST mutants begin at residue 21 and do not contain the cytosolic tail or transmembrane region (TM) (sPST and sPST140). Mutation of His³³¹ in PST to lysine (PST H331K) was previously shown to render a full-length PST protein catalytically inactive (57). The polybasic region (PBR) is found between residues 71 and 105 in PST. Mutation of Arg⁸² and Arg⁹³ in the PBR was previously shown to greatly reduce NCAM polysialylation (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).

FIGURE 2.

Expression of PST H331K, PST140, and sPST140 inhibits NCAM polysialylation by endogenous PST in SW2 cells. Myc-tagged PST, sPST, PST H331K, PST140, and sPST140 were transiently expressed in SW2 small cell lung carcinoma cells containing endogenous PST and polysialylated NCAM. Eighteen hours post-transfection, cells were fixed and indirect immunofluorescence was performed using the anti-Myc antibody to analyze enzyme localization (Myc, Localization) or the OL.28 anti-polySia antibody to analyze total polysialylation (OL.28, Polysialylation). Overlaying the fluorescent signals for PST protein localization and polysialylation demonstrated the impact that expressing exogenous PST proteins had on the polysialylation of endogenous SW2 cell NCAM (Myc/OL.28, Merge). Cells expressing exogenous PST proteins are marked with yellow arrowheads. Cells were examined using a Zeiss 200M inverted confocal microscope, ×63 oil immersion objective. Scale bar = 10 μm.

FIGURE 3.

NCAM polysialylation by endogenous PST is inhibited by expression of PST127 but not PST71 or PST62. Myc-tagged PST140, PST127, PST71, and PST62 were transiently expressed in SW2 small cell lung carcinoma cells. Eighteen hours post-transfection, cells were fixed and indirect immunofluorescence was performed to analyze enzyme localization (Myc, Localization) or total polysialylation (OL.28, Polysialylation). Overlaying the fluorescent signals for PST protein localization and polysialylation demonstrated the impact that expressing exogenous PST proteins had on the polysialylation of endogenous SW2 cell NCAM (Myc/OL.28, Merge). Cells expressing exogenous PST proteins are marked with yellow arrowheads. Cells were examined using a Zeiss 200M inverted confocal microscope, ×63 oil immersion objective. Scale bar = 10 μm.

FIGURE 4.

Wild type PST and all PST mutants containing the PBR effectively bind to NCAM, whereas PST71 and PST62 that lack the PBR do not. V5-tagged NCAM was co-expressed with Myc-tagged PST or a PST mutant in COS-1 cells (A) or Lec2 CHO cells (B). After 18 h, cells were lysed and lysates were incubated with anti-Myc antibody to immunoprecipitate (IP) the PST proteins, followed by incubation with protein A-Sepharose beads for 1 h. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting (IB) with anti-V5 antibody to detect co-immunoprecipitated NCAM (CoIP, IP: Anti-Myc antibody, IB: Anti-V5 antibody). To determine the relative expression levels of NCAM and PST proteins, an aliquot of lysate was removed prior to co-immunoprecipitation and subjected to SDS-PAGE and immunoblotting with anti-V5 antibody to detect NCAM (NCAM expression, Anti-V5 antibody) and anti-Myc antibody to detect the PST proteins (Enzyme expression, Anti-Myc antibody).

FIGURE 5.

Replacing Arg⁸² and Arg⁹³ block the ability of PST to enhance the polysialylation of SW2 cell NCAM and the ability of PST H331K to inhibit the polysialylation of SW2 cell NCAM by endogenous enzyme. Myc-tagged PST, PST R82A/R93A, PST H331K, PST H331K R82A, PST H331K R93A, and PST H331K R82A/R93A were transiently expressed in SW2 small cell lung carcinoma cells. Eighteen hours post-transfection, cells were fixed and indirect immunofluorescence was performed to analyze enzyme localization (Myc, green, Enzyme localization) or total polysialylation (OL.28, red, Polysialylation). Cells expressing exogenous PST and PST mutant proteins are marked with yellow arrowheads. Cells were examined using a Zeiss 200M inverted confocal microscope, ×63 oil immersion objective. Scale bar = 10 μm.

FIGURE 6.

The ability of PST H331K to inhibit the polysialylation of NCAM by endogenous SW2 PST is eliminated when Arg⁸² and Arg⁹³ are replaced with alanine residues. V5-tagged NCAM and Myc-tagged PST were expressed in COS-1 cells without (lane 1) or with Myc-tagged PST H331K, PST H331K R82A, PST H331K R93A, or PST H331K R82A/R93A in a 1:1:6 ratio (lanes 2–5). After 18 h, cells were lysed and NCAM was immunoprecipitated (IP) using the anti-V5 antibody. Immunoprecipitated NCAM was subjected to SDS-PAGE and immunoblotting (IB) to analyze NCAM polysialylation (NCAM polysialylation, IP: Anti-V5 antibody, IB: OL.28 Anti-polySia antibody). Relative NCAM, PST, and PST mutant protein expression levels were assessed by removing an aliquot of cell lysate prior to immunoprecipitation and subjecting it to SDS-PAGE and immunoblotting with anti-V5 antibody (NCAM Expression, Anti-V5 antibody) or anti-Myc antibody (Enzyme Expression, Anti-Myc antibody). Note that enzyme expression reflects the expression levels of both wild type and mutant PST proteins.

FIGURE 7.

The ability of PST to polysialylate NCAM is eliminated when Arg⁸² and Arg⁹³ are replaced, whereas the ability of PST to polysialylate NRP-2 and SynCAM 1 requires only the presence of Arg⁸². NCAM-V5 (A), NRP-2-V5 (B), or SynCAM 1-V5 (C) were co-expressed with Myc-tagged PST, PST R82A, PST R93A, or PST R82A/R93A in COS-1 cells. Eighteen hours post-transfection, cells were lysed and substrates were immunoprecipitated using the anti-V5 antibody. Immunoprecipitates (IP) were subjected to SDS-PAGE and immunoblotting (IB) with the OL.28 anti-polySia antibody to analyze the level of substrate polysialylation (Polysialylation, IP: Anti-V5 antibody, IB: OL.28 Anti-polySia antibody). Relative protein expression levels were determined by removing an aliquot of cell lysate prior to immunoprecipitation and subjecting it to SDS-PAGE followed by immunoblotting with anti-V5 antibody (NCAM, NRP-2, or SynCAM 1 Expression, Anti-V5 antibody) or anti-Myc antibody (Enzyme Expression, Anti-Myc antibody).