Piepkorn M, Hovingh P, Bennett K L, Aruffo A, Linker A
Department of Medicine, University of Washington School of Medicine, Seattle, WA, 98195-6524, USA.
Biochem J. 1997 Oct 15;327 ( Pt 2)(Pt 2):499-506. doi: 10.1042/bj3270499.
Previous studies have indicated that CD44 isoforms, spliced with variant exons, are heterogeneously glycanated with chondroitin sulphate and heparan sulphate chains. Because such alternative splicing may regulate divergent biological effects of the specific isoforms, we analysed the consequences of this process on the composition and structure of the chondroitin-sulphate chains. Recombinant chimaeras were engineered with and without exons V3-10 or V3,8-10 and expressed as Ig fusion proteins in COS cells. In addition, the chondroitin sulphates of wild-type isoforms were contrasted with those of isoforms mutated with serine-to-alanine codon substitutions at a putative Ser-Gly-Ser-Gly glycosaminoglycan acceptor site within exon V3. The chondroitin sulphates contained both 4- and 6-sulphated galactosamine residues, although there was a high content of non-sulphated galactosamine-containing repeat units. Splicing of exons V4-7, which contain no Ser-Gly consensus motifs, resulted in increased glycanation with chondroitin-sulphate chains, as well as increased sulphation levels of the polymers. Comparison of wild-type and acceptor-site mutant isoforms showed that chondroitin-sulphate content declined by more than 60-80% in the mutant, indicating that assembly of chondroitin-sulphate chains occurs there, and a general decrease in the sulphation level of the remaining chains was observed. Undersulphation of the recombinant chondroitin sulphates was shown by parallel analyses with native human keratinocyte CD44 molecules and is most probably an artifact of transient expression in COS cells. Our data indicate that combinatorial exon splicing exerts complex and distal effects on glycanation patterns and structure, which presumably modulate those functions that may be mediated though the chondroitin-sulphate moieties, such as motility and matrix invasion.
先前的研究表明，与可变外显子剪接的CD44亚型，会被硫酸软骨素和硫酸乙酰肝素链进行异质性糖基化。由于这种选择性剪接可能会调节特定亚型的不同生物学效应，我们分析了这一过程对硫酸软骨素链的组成和结构的影响。构建了含或不含外显子V3 - 10或V3,8 - 10的重组嵌合体，并在COS细胞中作为Ig融合蛋白表达。此外，将野生型亚型的硫酸软骨素与在外显子V3内假定的Ser - Gly - Ser - Gly糖胺聚糖受体位点处用丝氨酸到丙氨酸密码子替换进行突变的亚型的硫酸软骨素进行了对比。硫酸软骨素含有4 - 硫酸化和6 - 硫酸化的半乳糖胺残基，尽管含有非硫酸化半乳糖胺的重复单元含量很高。不含Ser - Gly共有基序的外显子V4 - 7的剪接，导致硫酸软骨素链的糖基化增加，以及聚合物的硫酸化水平增加。野生型和受体位点突变亚型的比较表明，突变体中硫酸软骨素含量下降了60 - 80%以上，这表明硫酸软骨素链的组装发生在该位点，并且观察到其余链的硫酸化水平普遍下降。通过与天然人角质形成细胞CD44分子的平行分析表明，重组硫酸软骨素存在硫酸化不足的情况，这很可能是COS细胞中瞬时表达的假象。我们的数据表明，组合外显子剪接对糖基化模式和结构产生复杂而深远的影响，这可能会调节那些可能通过硫酸软骨素部分介导的功能，如运动性和基质侵袭。
