Greenfield B, Wang W C, Marquardt H, Piepkorn M, Wolff E A, Aruffo A, Bennett K L
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA.
J Biol Chem. 1999 Jan 22;274(4):2511-7. doi: 10.1074/jbc.274.4.2511.
Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.
CD44的亚型被糖胺聚糖（GAGs）硫酸软骨素（CS）、硫酸乙酰肝素（HS）和硫酸角质素以不同方式修饰。GAG组装发生在丝氨酸之后紧跟甘氨酸（SG）的位点，但并非所有的SG位点都被利用。七个SG基序分布在五个CD44外显子中，在本文中我们确定了CD44中被利用的HS和CS组装位点。并非所有的CD44 SG位点都被修饰。CD44外显子V3中的SGSG基序是唯一的HS组装位点；该位点也被CS修饰。通过将V3基序中的丝氨酸突变为丙氨酸（AGAG），消除了该位点的HS和CS附着。外显子E5是CD44中另一个支持GAG组装并被CS修饰的外显子。我们在此使用多个重组CD44蛋白片段表明，V3中SGSG位点下游的八个氨基酸负责该位点HS的特异性添加。如果将CD44外显子E5中第一个SG位点下游的八个氨基酸与外显子V3中SGSG位点下游的那些氨基酸进行交换，E5中的SG位点就会被HS和CS修饰。同样，如果在E5中第一个SG下游发现的八个氨基酸置于V3中SGSG的下游，该位点会被CS修饰但不会被HS修饰。我们还表明，这些序列不能远距离指导CD44被HS修饰。含有将SGSG基序突变为AGAG的CD44外显子V3的构建体即使含有其他SG基序也不会被HS修饰。因此，已经确定了决定CD44上GAG合成的许多序列和结构要求。
