Abstract
Polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule (NCAM), is the key regulator of NCAM-mediated functions and crucial for normal brain development, postnatal growth, and survival. Two polysialyltransferases, ST8SiaII and ST8SiaIV, mediate polySia biosynthesis. To dissect the impact of each enzyme during postnatal brain development, we monitored the developmental changes in NCAM polysialylation in wild-type, ST8SiaII-, and ST8SiaIV-deficient mice using whole brain lysates obtained at 10 time points from postnatal days 1 to 21 and from adult mice. In wild-type and ST8SiaIV-null brain, polySia biosynthesis kept pace with the rapid increase in brain weight until day 9, and nearly all NCAM was polysialylated. Thereafter, polySia dropped by approximately 70% within 1 week, accompanied by the first occurrence of polySia-free NCAM-140 and NCAM-180. In ST8SiaII-null brain, polySia declined immediately after birth, leading to 60% less polySia at day 9 combined with the untimely appearance of polySia-free NCAM. Polysialyltransferase deficiency did not alter NCAM expression level or isoform pattern. In all three genotypes, NCAM-140 and NCAM-180 were expressed at constant levels from days 1 to 21 and provided the major polySia acceptors. By contrast, NCAM-120 first appeared at day 5, followed by a strong up-regulation inverse to the decrease in polySia. Together, we provide a comprehensive quantitative analysis of the developmental changes in polySia level, NCAM polysialylation status, and polysialyltransferase transcript levels and show that the predominant role of ST8SiaII during postnatal brain development is restricted to the first 15 days.
Highlights
Using delipidated whole brain homogenate, we determined the total amount of Polysialic acid (polySia) by DMB-HPLC analysis as described previously [35], and the results were displayed as relative polySia concentrations with the value obtained for postnatal day 1 (P1) wild-type brain set to 100% (Fig. 1A)
Within 8 days, the polySia level declined by almost 70% before a low but constant level was reached from P17 to P21
By making use of single knock-out mice lacking either ST8SiaII [31] or ST8SiaIV [32], we analyzed the impact of the two polysialyltransferases on polySia biosynthesis during postnatal brain development
Summary
Antibodies, Enzymes, and Reagent—NCAM-specific mouse monoclonal antibody (mAb) H28 [38] and polySia-specific mAb 735 [39] were used after affinity purification on protein Gand protein A-Sepharose (GE Healthcare), respectively. Protein Extraction and Western Blot Analysis—Mouse brains were homogenized [19], and one aliquot of each lysate was treated with 25 ng/l endosialidase for 45 min on ice. The proteins were separated by 7% SDS-PAGE under reducing conditions, loading 20 g of total protein/lane. RNA Extraction and Quantitative Real Time RT-PCR—RNA extraction and quantitative RT-PCR was performed as described [35] using a SDS7700 real time PCR system (Applied Biosystems) and the following gene-specific primers that span one intron: ST8SiaII, 5Ј-GGCTGTGGCCAGGAGATTG-3Ј and 5Ј-GGCATACTCCTGAACTGGAGCC-3Ј; ST8SiaIV, 5ЈGCACCAAGAGACGCAACTCATC-3Ј and 5Ј-CAGAGCTG-. DMB-HPLC Analysis—To analyze the total amount and the chain length pattern of polySia, the DMB-HPLC method [41] was used as described previously [35]. To determine the total amount of polySia, these peak areas were summarized
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