Structural Studies of the Endoglycosidase H-resistant Oligosaccharides Present on Human fi-Glucuronidase*

Human fi-glucuronidase bears 3-4 oligosaccharide moieties/subunit of Mr = 75,000. We have previously characterized the endoglycosidase H-releasable oligosaccharides of this enzyme including those which are phosphorylated and involved in targeting to lysosomes. In this study, we report the characterization of the endoglycosidase H-resistant oligosaccharides which were released from fi-glucuronidase with anhydrous hydrazine. Approximately 65% of the hydrazine-re-leased oligosaccharides are of the high mannose type, with the predominant species containing 9 mannose residues. The remaining oligosaccharides appear to originate from incomplete complex oligosaccharides.

Human fi-glucuronidase bears 3-4 oligosaccharide moieties/subunit of Mr = 75,000. We have previously characterized the endoglycosidase H-releasable oligosaccharides of this enzyme including those which are phosphorylated and involved in targeting to lysosomes. In this study, we report the characterization of the endoglycosidase H-resistant oligosaccharides which were released from fi-glucuronidase with anhydrous hydrazine. Approximately 65% of the hydrazine-released oligosaccharides are of the high mannose type, with the predominant species containing 9 mannose residues. The remaining oligosaccharides appear to originate from incomplete complex oligosaccharides. Their basic structures are Manal,6ManflB1,4Glc-NAcfal,4GlcNAcol, and Manal,3[Mana1,61Manf1,4Glc-NAci1,4GlcNAcol with roughly half of each species containing an additional fucose linked al1,6 to the Nacetylglucosaminitol (GlcNAcol) residue. The small amount of complex oligosaccharide present bearing 1 sialic acid was heterogeneous in nature with incompletion of the nonsialylated branch. In addition, there was a minor specie of high mannose-type oligosaccharide bearing 5 mannose residues with an al,6-linked fucose on the GlcNAcol. This structure was not expected since high mannose-type oligosaccharides have been reported to not be substrates for the al,6-fucosyl transferase.
Acid hydrolases are localized intracellularly within lysosomes. In the case of fibroblasts and presumably other cell types, transport of these hydrolases to the lysosomal compartment from their site of synthesis and/or from the extracellular milieu involves the specific recognition of high mannose-type oligosaccharides bearing 1 or 2 mannose-6-PO 4 moieties (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). In addition to receptor-mediated endocytosis based on recognition of mannose-6-P0 4 moieties, a receptor system is present on both alveolar macrophages and hepatic reticuloendothelial cells which is able to mediate the transport of acid hydrolases such as preputial ,-glucuronidase to lyso-* This study was supported in part by Biotechnology Mass Spectrometry Resource Grant PR00954 and by a special grant to the Department of Pathology from Brown and Williamson Tobacco Corp., Philip Morris, Inc., R.J. Reynolds Tobacco Co., and The United States Tobacco Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
f somes based on the recognition of high mannose-type oligosaccharides and specific degradation products of complex tetra-branched oligosaccharides (11,12). Although these distinct oligosaccharide-specific receptor systems have been extensively characterized, little information about the oligosaccharide structures present on lysosomal hydrolases has thus far been established. We have, therefore, examined the structures of all of the oligosaccharide species present on fi-glucuronidase prepared from human spleen (4). In a previous study, we reported the structural analysis of the phosphorylated and nonphosphorylated high mannose species released from ,Bglucuronidase by endoglycosidase H digestion (13). We report here the structures of the oligosaccharides subsequently released by treatment with anhydrous hydrazine.

DISCUSSION
Based on the mannose content/mol of fi-glucuronidase monomeric subunit and the proportion of each oligosaccharide specie found, the human ,-glucuronidase preparation examined contains 3-4 oligosaccharide moieties/subunit (Table II). Phosphorylated oligosaccharides account for 0.3 mol of oligosaccharide/mol of f-glucuronidase subunit, indicating that as much as 30% of the /f-glucuronidase monomeric species may bear a single phosphorylated oligosaccharide. Sinceglucuronidase is normally found in a tetrameric form, this would suggest that a high proportion of the tetramers contain at least 1 phosphorylated oligosaccharide moiety. It is also possible that some tetramers have multiple phosphorylated oligosaccharide moieties, while others contain no phosphate at all. Treatment of the native enzyme with endoglycosidase H results in the release of all the phosphorylated oligosaccharides and 1.0 additional mol of neutral high mannose-type oligosaccharide. The hydrazine-released oligosaccharides account for 2.0 mol of oligosaccharide/monomer. The high mannose oligosaccharides released by endoglycosidase H consist almost exclusively of species containing 5 and 6 mannose residues, whereas the dominant specie among the endoglycosidase H-resistant oligosaccharides contains 9 mannose residues (Table I).
Several interesting features of the hydrazine-released oligosaccharides are apparent from the structures and proportions shown in Table I. Although roughly half of the oligosaccharides released by hydrazine are of the high mannose type, 'Portions of this paper (including "Experimental Procedures," "Results," and Figs. 1-9) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-670, cite the authors, and include a check or money order for $8.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.   Manal --2Manal NANAa -Ga/l 4GlcNAc, -Manal 3 / aGlcNAcol, N-acetylglucosaminitol. those with 5, 6, 7, or 8 mannose residues proportion of the total, compared to the r mannose residues. Oligosaccharides with residues (M 2 , FM 2 , Ma, FM 3 , and Sl) m. from incompletely processed or degraded charides and account for a total of 0.78 mol mol of ,-glucuronidase subunit. The pr amount of fucosylated high mannose-ty with 5 mannose residues was unexpected Examination of the pathway for fucosylat gosaccharides has indicated that the high n narentlv can become fucosvlated under some circumstances. The presence of complex oligosaccharide moieties is connajor specie with 9 sistent with the binding of this and other lysosomal hydrolases 1, 2, or 3 mannose by Ricin agglutinin. In addition, secreted enzymes from I-cell iy have originated fibroblasts have a greater proportion of enzyme susceptible to complex oligosac-binding by Ricinus agglutinin (17). This may indicate that ofoligosaccharide/ inhibition of transport of these enzymes to the lysosome presence of a small permits the further processing of oligosaccharides with the pe oligosaccharide addition of peripheral galactose moieties. Thus, it is possible (FM 5 in Table I). that the B-glucuronidase we have characterized has incomtion of complex oli-pletely synthesized complex oligosaccharides rather than paraannose species are tially degraded ones. We cannot differentiate these possibili- It is clear from this study that, in addition to the phosphorylated oligosaccharides, ,-glucuronidase bears high mannose-type oligosaccharides which we have previously demonstrated can mediate the endocytosis of glycoproteins by reticuloendothelial cells (12). There are, however, no structures present which would be recognized by the hepatocyte Gal/ GalNAc-specific receptor (18). It may be that such structures are synthesized by further processing in patients with Mucolipidosis II or III and could account for the relatively normal enzyme levels found in the liver of these patients (19). Finally, it is not yet clear which of the phosphorylated high mannosetype oligosaccharides we and others have described are able to mediate transport to the lysosome. Studies with the individual characterized oligosaccharides will be required to answer this question. as subjected to hydratwnolysis. The enzyme oaa Suspended ij 0.4 L anhydrous hydrazine under a nitrogen atmosphere and heated in a sealed tube for S hours at 100'.

Human /i-Glucuronidase Oligosaccharides
The product eas eaporated under reduced pressure over concentrated ulfuric acid, then repeatedly eaporated with toluene to remove all hydrazine.
The oligosaccharides ere re-N-acetylated ith acetic anhydride in saturated MaHCO 3 . Following removal of N by passage over AGSO-XI2 (H), the product was subjected to descending chromatography on Whatman 3MM paper in btanol:ethanol:nater (4:1:1) for 48 hr to remove peptide degradation products.
The oligosaccharides were recovered by elation of the region from -2 to + c from the origin nith water.

NaB 3H4 Labeling of Hdrazine-released Oligosaccharides -
The hydrazine-released oligosaccharides were labeled ith a 10 fold molar ecess of NaB [3H] 4 in 1ml of .2M sodium borate. pH 9.8, at 30'C for hours. The reaction was terminated by addition of glacial acetic acid, and the product was passed oVer Downe AGSOW-X12 ().
The solution was dried and eaporated repeatedly with methanol to remove borate.
Chromatographic Methods -Bo6el P-4 (-400 mesh) i a l. r 100 cm column as tilized or the preparative fractionation of the oligosaccharide products. neutral oligosaccharides were subsequently analyzed n ioGel P-4 (21) or by high performance liquid chromatography on MktcroPk AX-S (30).
Sitalic acid bearing oligosaccharide species were analyzed by high erformance liquId chromatography on MicroPoR AX-IS (20). Analysis of sugar alditols was crried out as described by arr and Nordin (31).

RESULTS
Fractionation -The preparative fractionoation of the hydrazine released oligosaccharide species on Rioel P-4 is shown in Figure 1. The fractions were ooled as indicated and each neutrl fraction; i.e., -IX, was subsequently fractionated on MicroPak AX-S (Figure 2). The structure of each of the oligosaccharide species thus obtained was then determined.  The structure for Fraction IX is shown in Tble . The material eluting prior to fraction S1 consists of material smaller than monosaccharide.

Human fi-Glucuronidase Oligosaccharides
Fraction VIII -This fraction consisted of 2 species when examined on icroPak AX-5 (Fig. 2, Panel ). VIlla is identical in structure to IX ith the exception of an .1,6 linked fucose on the N-acetylglucosaninitol.
Intact Villa and the products of sequential digestion of Vlila with -mannosidase. o-mnnosidase and s-hexosainidase comigrated with the authentic fucosylated species (Fig. 4, Panels A, B and C). Treatent with o-fucosidase following removal of the 2 mannose residues converted the oligosaccharide to one which comigrated with authentic 62 (Fig.   4, Panel D). Periodate degradation also yielded G 2 rather than GlcNAc8l,4XylNAcol indicating that substituents are present at both positions 4 and 6 of the N-acetylglucosaminitol and that the 0-linked mannose is substituted at position 6 ( Figure 4, Panel E). These assignments were qualitatively confirmed by methylation which demonstrated the presence of Z,3,4,6-tetramethyl-mannose, 2,3,4-triethyl-mannose, and 3,4,6-trimethyl-fucose. The structure for Villa is shown in Table I.

GiG
The analyses for fraction Vlllb are summarized in Figure 5.
This material comigrated with authentic M 3 . Sequential digestion with a-mannosidase, -. annosidase and -hexosaminidase released 2 mannose, I mannose, and 1 N-acetylglucosamine residue, respectively (Fig. 5, Panels A, 8 and C). Unlike fractions IX and ViIla, Villb was susceptible to digestion with endoglycosidase (Panel D). Methylation demonstrated the presence of only 2,3,4,6-tetramethyl and 2,4-diethyl-anose confirming the structure shown in Table I Fraction VII -This fraction was also relatively homogeneous upon further fractionation on MicroPak AX-S (Fig. 2). As can be ascertained from the studies shown in Figure 6. this oligosaccharide differed from that in Fraction Villb only in the presence of an dditional el,6 linked fucose on the N-acetylglucsaminitol. This was confirmed by methylation analysis and by periodate oxidation (Fig. 6, Panel E) which yielded M 1 rather than Manls,4lcNAcl1,4XylNAcol indicating that the fucose was linked to the 6 position of the N-acetylglucosaminitol.
Fraction VI -This fraction proved to be heterogeneous when examined on MicroPak AX-5 (Fig. 2). The species designated Via, Vib, Ic and VId comigrated on both AX-5 and BioGel P-4 with authentic high mannose type oligosaccharides bearing 5, 6, 7 and 8 peripheral mannose residues respectively.
In each instance, the product of .-mannosidase (J.B.) digestion comigrated with authentic M 1 (Fig. 7, Panel A)  Fraction Via' was a new species of high mannose type oligosaccharide not previously encountered.
Analysis on BioGel P-4 yielded a mobility equivalent to 9.3 glucose equivalents which is roughly 0t. glucose equivalents larger than Via.
Digestion with o-annosidase yielded an oligosaccharide which comigrated with FMi (Fig. 7, Panel B) and could be further digested with -mannosidase and -hexosainidase to yield species which coigrated with 61cNAcS1,4 [Fucl,6] GlcNAol and Fucl,6GlcAcol (Fig. 7, Panel C). Digestion which endoglycosidase D also yielded a product which comigrated with Fucl,66lcNAcol (Fig. 7, Panel D), whereas digestion with endoglycoidase H yielded only the starting material. Digestion with a-fucosidase following 0-mannosidase and -mannosidase converted the product to one which comigrated with authentic G 2 . Digestion with A. satoi e1,2 mannosidase did not result in the release of any mannose. Thus, Via' appears to be a high mannose type oligosaccharide with an 1e,6 linked fucose on the asparaginelinked -acetylglucosamine. Fraction V -This was the major species among the hydrazine released oligosaccharides and was homogeneous by analysis on MicroPak AX-5 (Fig. 2).
Digestion with e1.2 specific annosidase from Aspergillus satoi yielded material of 8.5 glucose equivalents on BioGel P-4; i.e., coigrating with authentic 45 (Fig. 8, Panel C). ethylation analysis was also consistent with the structure shown in Table I   Fractions I and 1i -The major portion of the radiolabel in Fraction I and a part of that in Fraction II could not be identified as carbohydrate by gas chromatography or HPLC (31).
Further fractionation of fraction II on MicroPak AX-10 in 25 mM KPO 4 , pH 4.0 as described previously (13,20) yielded only material which corigrated with species having a single sialic acid moiety per oligosaccharide unit. Digestion with a-neuraminidase converted all of this material to neutral species. When eanined on ieGel P-4 following reoval of slalic acid, a heterogeneous product ranging from roughly 10 (o0) to 14 (914) glucose equivalents was seen (Fig. 9, Panel A). This was most consistent with a dibranched complex oligosaccharide with incompletion of I branch. Sequential digestion with o-galactosidse and -helosaminidase markedly reduced the heterogeneous appearance by shifting the larger species to material which comigrated with oligosaccharides in the range of 10 to 11 glucose equivalents (Fig. 9, Panels 8 and C). Following reoval of the sialic acid, digestion with -galactosidase and -hexosaminidase yielded species with mobilities on BinGel P-4 equal to 7.5 and 6.5 glucose equivalents (Fig. 9, Panel D). Digestion of this product ith endoglycosidase D converted 23 of the oligosaccharides to aterial which comigrated with Fucal,6cNAcol and the rest to GJcNAco) (Fig. 9, Panel F). Since digestion with o-galactosidase and n-heosaminidase followed by endoglycosidase O did not result in the production of GlcNAcol or Fucol,6GlcNAcol, it appears that essentially all of the sialic acid is present in the branch arising from the .1,3 linked mannose (32). In addition, the sensitivity of the product of 8-galactosidase and s-hexosawinidase digestion to a-mannosidase (Panel E), indicates the presence of a typical core structure for complex oligosaccharides with a linked annose residues,. Finally, digestion of the product shown in Pane) E with -fucosidase yielded only Z which migrates as 4 glucose equivalents (Fig. 9, Panel G) conf irming the presence of a-linked fucose.
The range of structures present in these oligosaccharide fractions is shown in Table 1. This material was present in such a small quantity as to prevent a ore detailed analysis of the heterogeneity. Analyses were performed on ioel P-4. The elation ositions of glucose oligoers used as standards re indicated as g,.

V
s the void voluIe and the included volume. Panel A; desialyoed fiction 11.