Sulfate contributes to the negative charge of podocalyxin, the major sialoglycoprotein of the glomerular filtration slits.

Abstract

Podocalyxin is the major sialoprotein ofthe rat glomerulus. Its function is to maintain the filtration slits of the glomerular epithelium open by virtue of its high net negative charge. We have used biosynthetic labeling and oligosaccharide analysis to characterize the anionic-charge-carrying moieties on this protein. Kidney slices from 2-day-old rats were biosynthetically labeled with [35SJCys, [3HJMan, [3H]GkcN, and 35SO4, after which podocalyxin was immunoprecipitated and purified by SDS/PAGE. AU these labels were incorporated into podocalyxin. Immunoprecipitates were subjected to digestion with specific glycosidases or digested with Pronase followed by chromatographic analysis of the released glycopeptides. Podocalyxin was susceptible to digestion with N-Glycanase and 0-Glycanase, indicating the presence of both N-and O-linked oligosaccharides. Approximately 30% of the [3H]GlcN-labeled glycopeptides bound to Con A, confirming the presence ofhigh mannose, hybrid, or biantennary N-linked structures; alkaline borohydride treatment confirmed the presence of O-linked oligosaccharides. Analysis of the 35SO4-labeled glycopeptides indicated that both the N-and O-linked structures were sulfated. We conclude that in newborn rat kidney (i) podocalyxin contains both 0-and N-linked oligosaccharides [high mannose or hybrid type, biantennary, and complex (sialylated) type], (i) podocalyxin is sulfated, and (iN) sulfate is located on both O-linked oligosaccharides and on glycopeptides carrying tri-or tetrantennary N-linked structures. These results indicate that the net negative charge of podocalyxin is most likely derived from sulfate as well as from sialic acid residues.The epithelium of the renal glomerulus is unique in that the intercellular spaces between interdigitating cell processes are normally open to allow passage of the glomerular filtrate, rather than being sealed off by typical tight junctions as is usually the case with epithelia in other locations. Maintenance of the patency of the intercellular spaces or filtration slits is dependent on the presence of highly negatively charged groups on the epithelial cell surface because, when the cell surface charge is neutralized by infusion of polycations, the filtration slits collapse and the adjoining cell membranes become closely opposed (1). Considerable evidence suggests that podocalyxin-a 140-kDa membrane glycoprotein present on the surfaces of the glomerular epithelium facing the urinary spaces (see Fig. 1)-plays a key role in maintaining the epithelial slits open. During development, the appearance of podocalyxin on the glomerular epithelium coincides with the opening of the intercellular spaces (2) and the disappearance of the tight junctions (3), which mark the transformation of this epithelium from a typical polarized epithelial cell layer to its adult arrangement.We have previously shown that podocalyxin is the major sialoprotein of the rat glomerulus and accounts for >50% of the total glomerular sialic acid content (4). Moreover, we found that the ...

Renal corpuscle of the sturgeon kidney: An ultrastructural, chemical dissection, and lectin‐binding study

“…In mammals, the podocytes have an unusual, highly negatively charged glycocalyx, which contains large amounts of podocalyxin (Kerjaschki et al, 1984). This molecule is a sialoprotein with a high affinity for WGA, and for PNA after neuramidase digestion (Dekan et al, 1991). In the sturgeon, however, the podocytes contain N-acetylglucosamine and lack sialic acid.…”

“…The negative charge of the filtration barrier depends on the presence of glycoconjugates in both the podocyte coat (Kerjaschki et al, 1984;Dekan et al, 1991) and the GBM (Kanwar and Farquhar, 1979;Kanwar et al, 1980). Glycoconjugates also appear to be involved in the maintenance of the podocyte morphology (Andrews, 1979).…”

Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells

“…Both were insensitive to endo H but were sensitive to sialidase digestion, yielding ϳ170-and ϳ95-kDa bands. This anomalous migration behavior resulting from loss of negative charge is characteristic of PC from rat kidney (Dekan et al, 1991) and is not unusual for heavily sialylated glycoproteins. After 20 min of chase, a 250-kDa band accumulated that most likely represents a dimer of PC.…”

“…The high apparent molecular mass compared with its predicted molecular weight is characteristic of heavily O-glycosylated glycoproteins. The 250-kDa band most likely represents a dimer of PC and is recognized by both monoclonal and polyclonal anti-PC (Dekan et al, 1991).…”

“…Immunoprecipitates were incubated with or without endo H (Streptomyces plicatus) or sialidase (Arthrobacter ureafaciens), separated by SDS-PAGE, and detected by fluorography. (Dekan et al, 1991), most likely represents a dimer of PC. A nonspecific, 160-kDa band (dot) that cross-reacts with anti-PC serum (0601) is seen in nontransfected and transfected MDCK cells.…”