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Glypican (Heparan Sulfate Proteoglycan) Is Palmitoylated, Deglycanated and Reglycanated During Recycling in Skin Fibroblasts

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Glypican (Heparan Sulfate Proteoglycan) Is Palmitoylated, Deglycanated and Reglycanated During Recycling in Skin Fibroblasts Glycobiology vol. 7 no. 1 pp. 103-112, 1997 Glypican (heparan sulfate proteoglycan) is palmitoylated, deglycanated and reglycanated during recycling in skin fibroblasts Gudrun Edgren1, Birgitta Havsmark, Mats Jonsson and granules (for reviews, see Kjell6n and Lindahl, 1991; Bernfield Lars-Ake Fransson et al., 1992; David, 1993; Heinegard and Oldberg, 1993). Pro- teoglycans are classified according to the characteristic fea- Department of Cell and Molecular Biology, Faculty of Medicine, Lund University, Lund, Sweden tures or properties of the core protein and can appear in many 'To whom correspondence should be addressed at: Department of Cell and glycoforms giving rise to considerable structural variation and Downloaded from https://academic.oup.com/glycob/article/7/1/103/725516 by guest on 30 September 2021 Molecular Biology 1, POB 94, S-221 00, Lund, Sweden functional diversity. In general, the protein part determines the destination of the proteoglycan and interacts with other mol- Skin fibroblasts treated with brefeldin A produce a recy- ecules at the final location. The glycan part provides the overall cling variant of glypican (a glycosylphosphatidylinositol- bulk properties as well as binding sites for other gly- anchored heparan-sulfate proteoglycan) that is resistant to cosaminoglycans and many types of proteins, including matrix inositol-specific phospholipase C and incorporates sulfate proteins, plasma proteins, enzymes, anti-proteinases, growth and glucosamine into heparan sulfate chains (Fransson, factors, and cytokines. L.-A. et aL, Glycobiology, 5, 407-415, 1995). We have now Cultured human fibroblasts synthesize, deposit, and secrete investigated structural modifications of recycling glypican, 3 a variety of proteoglycans and have been used extensively to such as fatty acylation from [ H]palmitate, and degrada- investigate both their biosynthesis and functional properties tion and assembly of heparan sulfate side chains. Most of 3 (see, e.g., Carlstedt et al., 1983; Lories et al., 1987; Heremans the H-radioactivity was recovered as lipid-like material et al., 1988; Schmidtchen et al., 1990a). Their plasma mem- after de-esterification. To distinguish between formation of brane-bound proteoglycans fall into two categories: those in- heparan sulfate at vacant sites, elongation of existing chains tercalated via membrane-spanning protein segments, for ex- or degradation followed by re-elongation of chain rem- ample, the syndecans, and those covalently linked to mem- nants, cells were pulse-labeled with [3H]glucosamine and 14 brane lipids of the phosphatidylinositol (Ptdlns)-type, so-called then chase-labeled with [ C]glucosamine. Material iso- glypiated proteoglycans (for review, see Bernfield et al., 1992; lated from the cells during the chase consisted of proteo- 3 Yanagishita and Hascall, 1992; David, 1993). Treatment of glycan and mostly [ H]-labeled heparan-sulfate degrada- fibroblasts with Ptdlns-specific phospholipase C releases a tion products (molecular mass, 20-80 kDa) showing that heparan-sulfate proteoglycan with a 60-70 kDa-core protein the side chains were degraded during recycling. The de- (David et al., 1990; Schmidtchen et al., 1990b) which, after gradation products were initially glucuronate-rich, but be- molecular cloning of its cDNA, was given the name glypican came more iduronate-rich with time. The glypican proteo- (David et al., 1990). glycan formed during the chase was degraded either with Glycosyl-Ptdlns-anchored membrane proteins are fairly alkali to release intact side chains or with heparinase to common and include a variety of different protein families (for generate distally located chain fragments that were sepa- reviews, see Englund, 1993; Udenfriend and Kodukula, 1995). rated from the core protein, containing the proximally lo- Proteoglycans linked in this manner include the above- cated, covalently attached chain remnants. All of the [ C]- mentioned glypican with a 64 kDa core protein and found in radioactivity incorporated during the pulse was found in many different cell types (for review, see David, 1993) and peripheral chain fragments, and the chains formed were K-glypican with a 57.5 kDa core protein (Watanabe et al., not significantly longer than the original ones. We there- 1995) as well as other closely related forms, such as cerebro- fore conclude that newly made heparan-sulfate chains were glycan (Karthikeyan et al, 1992; Stipp et al., 1994), one spe- neither made on vacant sites, nor by extension of existing cies attached to muscle cells (Campos et al., 1993), another chains but rather by re-elongation of degraded chain rem- with a 39 kDa core protein isolated from adipocytes (Misra et nants. The remodeled chains made during recycling ap- al., 1994), and a developmentally regulated intestinal form, peared to be more extensively modified than the original named OCI-5 (Filmus et al., 1995). The exact function of the ones. lipid anchor is not clear. In polarized cells, it may serve to Key words: fatty acylation/glypican/heparan sulfate/recycling/ direct proteins and proteoglycans to the apical surface. In other reglycanation cases, glypiated products may be directed and concentrated to specific membrane patches, called caveolae (Anderson, 1993). Biosynthesis and turn-over of glypican have been studied in Introduction granulosa cells (see Yanagishita, 1992; Yanagishita and Has- Proteoglycans are special forms of glycoproteins that are co- call, 1992). In these cells, glypican and other heparan sulfate- valently substituted with linear and sulfated glycosaminogly- proteoglycans are segregated and directed to separate degrada- cans (keratan sulfate, chondroitin sulfate, dennatan sulfate, tion pathways. Glypican seems to be exclusively internalized heparan sulfate, or heparin or combinations thereof). They and degraded. However, in a parathyroid cell line, Takeuchi et have a wide tissue distribution and occur in various forms of al. (1990) demonstrated recycling of heparan sulfate proteo- extracellular matrices, at cell surfaces and in intracellullar glycan between the cell surface and an intracellular compart- © Oxford University Press 103 G.Edgren et aL ment. Partial endoglycosidic degradation of heparan sulfate by endo-P-glucuronidase has been detected in many types of cells (for references, see Schmidtchen and Fransson, 1994). The core proteins of proteoglycans are synthesized on mem- brane-bound ribosomes in the endoplasmic reticulum, and then transported to the Golgi, where glycan side chains are as- sembled. Mature proteoglycans are either secreted into the ex- tracellular space or retained at the cell surface. By using brefel- din A to block transport from the endoplasmic reticulum to the Golgi, we have previously shown that most of the proteogly- cans produced by skin fibroblasts are derived from newly syn- thesized core proteins (Fransson et aL, 1992). However, a por- tion (-20%) of cell surface-bound heparan sulfate proteoglycan can be metabolically labeled with both radiosulfate and radio- active carbohydrate precursors in the presence of brefeldin A. Downloaded from https://academic.oup.com/glycob/article/7/1/103/725516 by guest on 30 September 2021 In the presence of suramin, which blocks internalization and deglycanation of proteoglycans, skin fibroblasts accumulated a membrane-bound heparan sulfate proteoglycan with a 60-70 kDa core protein (Fransson et aL, 1995). When both drugs were used simultaneously, no radiolabeled proteoglycan was detected suggesting that the radiolabeled proteoglycan was de- rived from resident cell-surface proteoglycan. After chemical biotinylation of cell-surface proteoglycan followed by meta- bolic radiosulphation, in continuously brefeldin A—treated cells, biotin-tagged radiolabeled proteoglycan was demon- strated, indicating the presence of recycling proteoglycan spe- cies. To determine the nature of the core protein, fibroblasts were pulse-labeled with [3H]leucine or [3H]inositol in the pres- ence of suramin, followed by chase-labeling with [35S]sulfate in the presence of brefeldin A. A hydrophobic, glycosyl- Ptdlns-anchored, heparan sulfate proteoglycan with a 60-^65 kDa core protein was obtained, indicating that it was glypican. 4 6 8 12 However, the proteoglycan was resistant to digestion with Pt- Fraction number dlns-specific phospholipase C (Fransson et al., 1995), which could be due to fatty acylation of the inositol moiety. Fig. 1. Chromatography of [3H]palmitate (O) - and [33S]sulfate (•^labeled proteoglycan on (A) Superose 6, (B) Mono Q, and (C) octyl-Sepharose. In the present study we have investigated whether the recy- Cells grown to confluence in 25-cm2 dishes were metabolically radiolabeled cling glypican variant can be fatty acylated, and whether the in a sulfate-deficient medium containing 1% (v/v) donor calf serum, 50 u,Ci/ml [33S]sulfate, 20-50 jtCi/ml [3H]palmitate, and brefeldin A (10 heparan sulfate side chains are built on vacant sites, if existing (ig/ml) at 37°C for 24 h (Masterson and Magee, 1992). The medium was chains are directly elongated or if they are degraded and re- removed and proteoglycan was recovered from a Triton X-100 extract of elongated. the cells by chromatography on DEAE-cellulose as described in Materials and methods. The proteoglycans were first chromatographed on Superose 6 in 4 M guanidinium chloride, pooled (fractions 20-29 in A), dialyzed against low-ionic strength buffer, chromatographed on MonoQ eluted with a NaCl gradient going from 0.15 M (fraction 10) to 1.2 M (fraction
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