Identification of Endoglycan, a Member of the CD34/Podocalyxin Family of Sialomucins

Abstract

CD34 and podocalyxin are structurally related sialomucins, which are expressed in multiple tissues including vascular endothelium and hematopoietic progenitors. These glycoproteins have been proposed to be involved in processes as diverse as glomerular filtration, inhibition of stem cell differentiation, and leukocyteendothelial adhesion. Using homologies present in the cytoplasmic tails of these proteins, we have identified a novel member of this family, which we designate endoglycan. This protein shares a similar overall domain structure with the other family members including a sialomucin domain, but also possesses an extremely acidic amino-terminal region. In addition, endoglycan contains several potential glycosaminoglycan attachment sites and is modified with chondroitin sulfate. Endoglycan mRNA and protein were detected in both endothelial cells and CD34؉ bone marrow cells. Thus, CD34, podocalyxin, and endoglycan comprise a family of sialomucins sharing both structural similarity and sequence homology, which are expressed by both endothelium and multipotent hematopoietic progenitors. While the members of this family may perform overlapping functions at these sites, the unique structural features of endoglycan suggest distinct functions for this molecule.CD34 and podocalyxin are evolutionarily related glycoproteins, which share a similar overall domain structure as well as significant sequence homology. Structurally, the extracellular region of each of these molecules is dominated by an aminoterminal mucin-like domain, which is densely substituted with sialylated O-linked carbohydrates. This extensive glycosylation causes mucin domains to adopt an extended, rodlike structure (1, 2). The mucin-like region is followed by a cysteine-containing and presumably globular domain. This domain may fold into an immunoglobulin-like structure as the positions of 2 of the cysteines are conserved in the C2 set of the immunoglobulin superfamily (3). The cytoplasmic domains of these proteins are 73-76 amino acids in length and highly conserved between species orthologs. It is also in this region that the highest homology between CD34 and podocalyxin is found (4).

Endoglycan, a Member of the CD34 Family, Functions as an L-selectin Ligand through Modification with Tyrosine Sulfation and Sialyl Lewis x

“…Endoglycan cDNA Constructs-The generation of the EG/IgG and AD/IgG expression constructs in vector pEF-BOS has been described (33). The AD/IgG mutants YYA and FYT were generated by site-directed mutagenesis following standard protocols.…”

“…Cells were maintained for 10 days in selection medium supplemented with puromycin. Transfectants expressing high levels of endoglycan were determined by flow cytometry analysis employing an endoglycan-specific antibody generated against a GST fusion protein of endoglycan (33). For 35 SO 4 labeling, cells were grown to ϳ70% confluence in a 175 cm 2 flask and cultured as described above with 2 mCi of Na 2 35 SO 4 .…”

“…Endoglycan, like CD34 and podocalyxin, is widely expressed. Its distribution includes vascular endothelium, smooth muscle, hematopoietic precursor populations and leukocyte subpopulations (33). Here, we demonstrate that endoglycan with appropriate post-translational modifications interacts with L-selectin through recognition of sLex and tyrosine sulfates in the amino-terminal region of the molecule.…”

“…We therefore employed an Fc fusion protein containing just the amino-terminal domain of the molecule (AD/IgG) (33). Recombinant EG/IgG and AD/IgG chimeras were produced in the presence of exogenously provided FTVII and used as substrates in flow chamber experiments as above.…”

“…Endoglycan was originally cloned based on sequence homologies in its cytoplasmic domain to CD34 and podocalyxin (33). It shares a similar overall structure with these molecules, including a mucin-like domain and a membrane proximal globular domain.…”