Drosophila heparan sulfate, a novel design

Abstract

Heparan sulfate (HS) proteoglycans play critical roles in a wide variety of biological processes such as growth factor signaling, cell adhesion, wound healing, and tumor metastasis. Functionally important interactions between HS and a variety of proteins depend on specific structural features within the HS chains. The fruit fly (Drosophila melanogaster) is frequently applied as a model organism to study HS function in development. Previous structural studies of Drosophila HS have been restricted to disaccharide composition, without regard to the arrangement of saccharide domains typically found in vertebrate HS. Here, we biochemically characterized Drosophila HS by selective depolymerization with nitrous acid. Analysis of the generated saccharide products revealed a novel HS design, involving a peripheral, extended, presumably single, N-sulfated domain linked to an N-acetylated sequence contiguous with the linkage to core protein. The N-sulfated domain may be envisaged as a heparin structure of unusually low O-sulfate content.

FIGURE 1.

Structure of HS chain and approaches for analysis. A, the structural domains (NS-, NA/NS-, and NA-domains) are defined with regard to the distribution of GlcNS and GlcNAc units. B, schematic representation of methods used to elucidate HS domain structure. For further information, see “Results” and “Experimental Procedures.” The symbols used are defined below the scheme. GlcA, GlcUA; IdoA, IdoUA.

FIGURE 2.

Gel chromatography of ³H-labeled saccharides obtained after deaminative cleavage of HS. HS samples from Drosophila or pig intestinal mucosa were deaminated and radiolabeled according to Protocol A (A and B) or Protocol B (C and D) (see “Experimental Procedures,” Fig. 1, and supplemental Fig. S1), and the products were analyzed by Bio-Gel P-10 gel chromatography. The numbers above each peak in A indicate oligosaccharide size.

FIGURE 3.

High-voltage paper electrophoresis of ³H-labeled disaccharides. ³H-Labeled disaccharides isolated by gel chromatography (Sephadex G-15) after deamination and radiolabeling of Drosophila HS according to Protocol A (A) or Protocol B (B) (see “Experimental Procedures,” Fig. 1, and supplemental Fig. S1) were fractionated by high-voltage electrophoresis on Whatman No. 3MM paper at pH 5.3. After drying, papers were cut into 1-cm segments and analyzed for radioactivity. Arrows indicate the migration positions of non-sulfated (1), mono-O-sulfated (2), and di-O-sulfated (3) HexUA-aMan_R disaccharide standards.

FIGURE 4.

Anion-exchange HPLC of ³H-labeled disaccharides. Disaccharides generated by deamination/radiolabeling according to Protocol A (A) or Protocol B (B) were analyzed by anion-exchange HPLC on a Partisil 10 SAX column. C, end-labeled, N-sulfated oligosaccharides obtained through Protocol B were deaminated at pH 1.5, and the released labeled disaccharide was isolated and subjected to anion-exchange HPLC. (Note that the [³H]aMan_R residues here represent a GlcNAc unit in the intact HS chains; the corresponding reaction is illustrated to the right.) The dashed line in A refers to a sample digested with liver β-glucuronidase/α-iduronidase prior to chromatography. Arrows indicate the elution positions of disaccharide standards (with the corresponding disaccharide units in the intact HS chain shown in parentheses): 1,2, GlcUA-aMan_R, IdoUA-aMan_R (-GlcUA/IdoUA-GlcNAc/NS-), aMan_R, and aMan_R6S (generated by β-glucuronidase/α-iduronidase digestion of disaccharides) (peaks not individually identified); 3, GlcUA-aMan_R6S (-GlcUA-GlcNS6S-); 4, IdoUA-aMan_R6S (-IdoUA-GlcNS6S-); 5, IdoUA2S-aMan_R (-IdoUA2S-GlcNS-); 6, IdoUA2S-aMan_R6S (-IdoUA2S-GlcNS6S-). Note that only the aMan_R residues indicated by open triangles were radiolabeled (derived from GlcNAc residues in the intact polysaccharide).

FIGURE 5.

Schematic presentation of HS/heparin chains. GlcA, GlcUA; IdoA, IdoUA.