Characterization of the Heparan Sulfate and Chondroitin Sulfate Assembly Sites in CD44

Abstract

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.

Heparan Sulphation Patterns Generated by Specific Heparan Sulfotransferase Enzymes Direct Distinct Aspects of Retinal Axon Guidance at the Optic Chiasm

“…CD44 is confined to more central regions of the neuronal array than SSEA-1 and has been shown to be required for axon crossing of the midline (Sretavan et al, 1995; Lin and Chan, 2003). Different isoforms of CD44 exist and are decorated with various sulfated glycosamino- glycans (Brown et al, 1991;Piepkorn et al, 1997;Greenfield et al, 1999), and HS immunoreactivity overlaps with SSEA-1-expressing chiasm neurons (Chung et al, 2001). Hs2st and/or Hs6st1 enzymes may normally sulfate SSEA-1 and/or CD44 and influence the responsiveness of the growth cone to HSPGdependent guidance cues such as Slit2 (Piper et al, 2006) or fibroblast growth factor (Ornitz, 2000).…”

Heparin and Heparan Sulfate Biosynthesis

“…One such example is thrombomodulin, which may bear a CS chain (30) or a linkage tetrasaccharide (31). Proteoglycans such as serglycin, betaglycan, and CD44 can be glycanated at a particular tetrasaccharide attachment site by either CS or HS (3,32). Agrin, which triggers the local aggregation of proteins at the developing neuromuscular junctions and was recently found on T lymphocytes (33), may be another example.…”

A mutation in a CD44 variant of inflammatory cells enhances the mitogenic interaction of FGF with its receptor

“…Heparin-binding growth factors such as FGF-2 can be bound to the V3-associated HS of v3-containing CD44 variants (but not to other variant exons) and then presented autocrinally or paracrinally to the corresponding receptors (30). Flow cytometry revealed that PE-labeled FGF-2 bound at a wide range of concentrations (0.1-20 nM) (not shown) at an extent similar to Namalwa-CD44v3-v10 cells and to Namalwa-CD44vRA cells (Figure 3; 10 nM FGF-2), both in the absence (Figure 3a, inset) and in the presence (Figures 3, a and b) of 0.2 M NaCl, which reduces the nonspecific binding of FGF-2.…”

“…It was found, however, that the assembly of HS is determined by eight amino acids located downstream from the exon v3 SGSG motif. Because exon v3 of the CD44 proteoglycan is the only one containing this sequence in the context of the SG or SGSG motif, HS assembly is confined to this exon (30). The sequestering of HS-binding growth factors on the v3 exon of CD44 proteoglycan has been well documented (5)(6)(7)(8)(9)(10)(11)(12).…”

“…The proteoglycan nature of CD44 of cell surfaces is well established (8,9,30). CD44 includes seven potential consensus single serine-glycine (SG) or double SGSG assembly sites for glycosaminoglycans (GAGs), such as HS or chondroitin sulfate.…”