Endothelial Heparan Sulfate Proteoglycans That Bind to L-Selectin Have Glucosamine Residues with Unsubstituted Amino Groups

Abstract

We earlier reported calcium-dependent, heparin-like L-selectin ligands in cultured bovine endothelial cells (Norgard-Sumnicht, K. E., Varki, N. M., and Varki, A. (1993) Science 261,480-483). Here we show that these are heparan sulfate proteoglycans (HSPGs) associated either with the cultured cells or secreted into the medium and extracellular matrix. Activation of the endothelial cells with bacterial lipopolysaccharide (LPS) does not markedly alter the amount or distribution of this material. A major portion of the glycosaminoglycan (GAG) chains released from these HSPGs by alkaline beta-elimination rebinds to L-selectin in the presence of calcium, indicating that these saccharides alone can mediate the high affinity recognition. Heparin lyase digestions indicate that these GAG chains are enriched in heparan sulfate, not heparin sequences. Current understanding of the biosynthesis of heparan sulfate chains indicates that all glucosamine amino groups must be either N-acetylated or N-sulfated. However, nitrous acid deamination at pH 4.0 suggests the presence of some unsubstituted amino groups in these L-selectin-binding GAG chains from endothelial cell HSPGs. This is confirmed by chemical N-reacetylation and by reactivity with sulfo-N-hydroxysuccinimide-biotin. These unsubstituted amino groups are also found on HSPGs from human umbilical vein endothelial cells, but are not detected in those from Chinese hamster ovary cells. In both bovine and human endothelial cells, these novel groups are enriched for in the HS-GAG chains which bind to L-selectin. Despite this, studies with N-reacetylation and nitrous acid deamination do not show conclusive evidence for the direct involvement of the unsubstituted amino groups in L-selectin binding. This may be because the chemical reactions used to modify the amino groups do not go to completion. Alternatively, the unsubstituted amino groups may only be indirectly involved in generating binding, by dictating the biosynthesis of another critical group. Regardless, these studies shown that HSPGs from cultured endothelial cells which can bind to L-selectin are enriched with unsubstituted amino groups on their GAG chains. The possible biochemical mechanisms for generation of these novel groups are discussed.

Location of N-Unsubstituted Glucosamine Residues in Heparan Sulfate

“…The quantity of GlcNH 2 was indicated by the level of 3 H incorporation achieved following HNO 2 -pH 3.9 treatment of HS. The results ranged from 0.7 to 4% of total disaccharide units in three different samples, in fair agreement with previous reports based on other techniques (18,19,26). The specificity of our approach was ascertained by chemical N-acetylation of parent polysaccharide samples, which were found to preclude 3 H incorporation.…”

“…A monoclonal antibody that recognized GlcNH 2 units in HS thus bound in selective fashion to extracellular tissue components in fresh-frozen rat kidney (18). The presence of GlcNH 2 residues was found to correlate with the ability of bovine and human endothelial HS to bind L-selectin (although binding was not critically dependent on the GlcNH 2 residues) (19). Moreover, GlcNH 2 units were identified as targets for a 3-O-sulfotransferase isoform (3-OST-3A) that introduces a sulfate substituent at C3 (20,21) and thereby * This work was supported by grants from the Swedish Foundation for Strategic Research, Grant QLK-CT-1999.00536 from the European Commission program, and from the Polysackaridforskning AB (Uppsala).…”

Multiple Isozymes of Heparan Sulfate/Heparin GlcNAcN-Deacetylase/GlcN N-Sulfotransferase

“…NDST3) but left unmodified. Heparan sulfates and heparin are known to contain a small proportion of N-deacetylated glucosamine units (33)(34)(35), and some of these units serve as substrates for selective modifying enzymes, such as the 3-O-sulfotransferases (36,37). Additional studies with defined, chemically modified substrates are needed to determine which of these possibilities are correct.…”

Heparan Sulfate Proteoglycans

“…To circumvent this problem, partially cleaved preparations were employed, eventually leading to the identification of minimally sized oligosaccharides that bound with reasonable affinity and that in some cases would initiate a biological response. Examples include, but are not limited to, FGF2 (Guimond et al 1993;), platelet-derived growth factor (Feyzi et al 1997), platelet factor 4 Stringer and Gallagher 1997), MIP1a (Stringer et al 2002(Stringer et al , 2003, hepatocyte growth factor (scatter factor) (Lyon et al 1994;Ashikari et al 1995), vascular endothelial growth factor (Soker et al 1994;Ono et al 1999;Ashikari-Hada et al 2005;Robinson et al 2006), lipoprotein lipase (Parthasarathy et al 1994;Spillmann et al 2006), amyloid , and L-selectin (Norgard-Sumnicht and Varki 1995;Wang et al 2002). When FGF1 was studied in detail, it was noted that a range of HS octasaccharides that varied in the number as well as the positions of individual sulfate groups could bind with varying affinity (Kreuger et al 2001).…”