Structural Features of Tissue Glycoproteins. Fractionation and Methylation Analysis of Glycopeptides Derived from Rat Brain, Kidney and Liver

Eur. J. Biochem. 78, 369-379 (1977)

Structural Features of Tissue Glycoproteins Fractionation and Methylation Analysis of Glycopeptides Derived from Rat Brain, Kidney and Liver

Tom KRUSIUS and Jukka FINNE

Department of Medical Chemistry, University of Helsinki

(Received March 18, 1977)

1. Glycopeptides were prepared by papain digestion from delipidated brain, kidney and liver and fractionated by the combination of concanavalin-A-affinity chromatography, and gel filtration after prior treatment with NaOH/NaBH, . All tissues contained four fractions of structurally distinct carbohydrate chains : 0-glycosidically-linked oligosaccharides, two different types of acidic N-glycosidically-linked oligosaccharides (fractions A and B) and mannose-rich N-glycosidically-linked oligosaccharides (fraction C). The relative amounts of these fractions varied in different tissues. The 0-glycosidically-linked oligosaccharides accounted for 3 - 9%, fraction A glycopeptides for 50 - 60%, fraction B glycopeptides for 21 - 34% and fraction C glycopeptides for 21 - 34% of total glycopeptide carbohydrate.

2. The 0-glycosidically-linked oligosaccharides are mainly composed of fi-galactosyl(1- 3)-N- acetylgalactosamine and its monosialosyl and disialosyl derivatives. No large molecular size O-glycosidically-linked carbohydrate chains similar to those present in mucus of the digestive tract and ovarian cysts were detected. Fraction A and B glycopeptides resemble by their carbohydrate composition and substitution pattern of their sugar components the N-glycosidically-linked oligosaccharides of fetuin and transferrin, which behave similarly on concanavalin A-affinity chromatography, respectively. However, the results of the methylation analysis suggest that considerable heterogeneity occurs at the mannose branch-points of fraction A and B glycopeptides as compared to the reference compounds. Fraction C contained only mannose-rich glycopeptides with a structure similar to those of ovalbumin and thyroglobulin type-A glycopeptides.

3. Methylation analysis showed clear differences in the substitution pattern of galactose and N-acetylglucosamine, which together with N-acetylneuraminic acid are supposed to form the peripheral branches of fraction A and B glycopeptides. Brain glycopeptides contained relatively high amounts of terminal nonsubstituted galactose and N-acetylglucosamine suggesting that the peripheral branches often are incomplete. N-Acetylneuraminic acid was mainly attached to the C-3 of the galactose residues in brain glycopeptides. Kidney glycopeptides contained less terminal nonsubstituted N-acetylglucosamine and proportionally more galactose substituted at C-6 than brain gly-. copeptides. In contrast to brain and kidney, no terminal N-acetylglucosamine and only small amounts of terminal nonsubstituted galactose occurred in liver suggesting that the peripheral branches are mainly complete. In liver galactose was substituted nearly as often at C-6 as at C-3.

4. Considerable differences occurred in the relative amount of fucose from one tissue to another. Brain glycopeptides contained fucose 2 and 4 times more than kidney and liver glycopeptides, respectively. Fucose was mainly bound to C-3 and C-6 of N-acetylglucosamine and not to galactose as proposed generally.

5. Based on the results of the present study it may be concluded that brain, kidney and liver glycoproteins contain four structurally distinct types of carbohydrate chains resembling each other. However, the structure of the terminal parts of the carbohydrate chains are glycosylated in a pattern characteristic to each tissue.

Interactions between the cell and its environment . . ~ ~ .._._. ..- .~~ ~~~~~~~~~~

Abbreviations. AcNeu, N-acetylneuraminic acid; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine;

are important in cell growth, development and main- Man, mannose. tenance of multicellular organisms. The carbohydrate

Enzymes. Vibrio cholerae neuraminidase, (EC 3.2.1.18); a-L-fu- chains of membrane dycoproteins are supposed to cosidase (EC 3.2.1.51). take part in these specific interactions [l]. Since cells

370 Carbohydrate Chains of Tissue Glycoproteins

derived from different tissues have specific adhesive- ness, it is possible that the structure of carbohydrate chains in membrane glycoproteins differs from one tissue to another. With the exception of the O-glycosidically-linked oligosaccharides no comparative stud- ies on the structure of carbohydrate chains derived from tissue glycoproteins have been described.

Tissue glycoproteins are for their most part bound to membranes. In brain only about 20% of protein bound carbohydrate is soluble [2]. Attempts to purify detergent-solubilized glycoproteins for structural analysis have not been successful. In contrast, carbohydrate chains of membrane glycoproteins are nearly quantitatively solubilized in the form of glycopeptides by proteolytic enzymes [3]. Difficulties in the fractionation and purification of these glycopeptides has precluded accurate structure analysis of the carbohydrate chains of tissue glycoproteins.

In the present communication we describe a general method which makes possible the isolation of fractions containing structurally distinct glycopeptides. Using this fractionation method it was shown that the glycoproteins of brain, kidney and liver contain four different types of carbohydrate chains: 0-glycosidically-linked oligosaccharides, two structurally different types of acidic N-glycosidically- linked oligosaccharides and mannose-rich N-glycosidically-linked oligosaccharides. Tissue glycoproteins thus structurally resemble known soluble glycoproteins. However, the amount of different types of carbohydrate chains varied from one tissue to another. Furthermore, methylation analysis showed tissue- specific differences in the substitution pattern of sugar residues located in the peripheral branches of the carbohydrate chains. Such structural differences may be important for the specificity in the interactions of the cell and its environment, such as cell recognition and adhesion.

MATERIALS AND METHODS

Preparation of Glycopeptides

Adult albino Wistar rats were used. Whole brains, kidneys and livers were delipidated by homogenization in chloroform/methanol [4]. Glycopeptides were prepared by papain digestion and purified by gel filtration on Sephadex G-25 as described previously [4]. Glyco- peptides from liver were also purified by gel filtration on Sephadex G-50 (2 cmx75 cm) to remove high- molecular-weight glucose polymers [4].

Fractionation of Glycopeptides on Concanavalin A ISepharose

The purified glycopeptides were fractionated by affinity chromatography on concanavalin A/Sepharose

(1.3 cm x 8 cm) by step-wise elution with a-methyl-D- glucoside as described previously [5]. Glycopeptides not bound by concanavalin A were eluted with the starting buffer, 5 mM sodium acetate (pH 5.2) containing 0.1 M NaCl and CaCl,, MnCl, and MgC1, (1 mM each). Glycopeptides interacting weakly with the lectin were obtained by elution with 20 mM a-methyl-D-glucoside and glycopeptides interacting strongly with the lectin by elution with 200 mM a- methyl-D-glucoside, both in the starting buffer.

Separation of N-Glycosidic and 0-Glycosidic Carbohydrate Chains

Glycopeptides were treated with 0.05 M NaOH in 1 M NaBH, for 16 ha t 45 "C [6]. NaBH, was destroyed with glacial acetic acid and the solution was desalted on a column (3 cm x 70 cm) of Sephadex G-10, eluted with 0.1 M pyridine/acetic acid buffer (pH 5.0) [7]. The released oligosaccharides were separated from the N-glycosidic glycopeptides on Sephadex G-50 (2 cm x 75 cm), eluted with 0.1 M pyridine/acetic acid buffer (pH 5.0).

Digestion with Glycosidases

Glycopeptides containing about 400 nmol of N-acetyl-neuraminic acid were treated with 5 U Vibrio cholerae neuraminidase in 0.2 ml of 10 mM Tris-acetate buffer (pH 6.8) containing 2 mM CaC1, at 37 "C for 24 h, after which 5 U of neuraminidase was added and the incubation was continued for another 24 h. Fucosidase treatment was carried out with glycopeptides containing about 150 nmol of fucose in 0.15 ml of 0.2 M sodium acetate buffer (pH 4.5) and 0.5 U a-L-fucosidase from beef kidney at 37 "C for 24 h, after which 0.5 U of a-L-fucosidase was added and the incubation was continued for another 24 h.

Subcellular Fractionation of Brain and Liver

The microsomal and soluble fractions of rat liver were prepared from a 1 : 10 homogenate in 0.25 M sucrose. Large particles were removed by centrifuga- tion at 10000 x g for 10 min and the microsomes were sedimented at 100 000 x g for 60 min. The microsomes were homogenized in 10 mM sodium phosphate buffer (pH 7.1) in order to liberate entrapped soluble glycoproteins, and resedimented as above. The microsomal and soluble fractions of rat brain were prepared as described by Whittaker [8]. The soluble fractions were dialyzed against distilled water, lyophilized and sus- pended in a small volume of 0.15 M NaCl. Microsomes and the suspensions were delipidated with chloroform/ methanol [4].

T. Krusius and J. Finne 371

Analytical Methods

Monosaccharides were determined by gas-liquid chromatography [9]. Neuraminic acid in the column effluents was determined colorimetrically [lo, 111. The core disaccharide of the alkali-labile 0-glycosidic carbohydrate chains (~-galactosyl-(l-3)-N-acetylgalactosaminitol) was determined after mild acid hydrolysis as its trimethylsilyl derivative by gas-liquid chromatography and mass spectrometry [7].

Glycopeptides were methylated by the method of Hakomori [12] and degraded as described by Stellner et al. [13]. Partially methylated alditol acetates were analyzed by gas-liquid chromatography and mass spectrometry. Alditol acetates of neutral sugars except monomethylated hexoses were chromatographed on 1 % OV-225 or 3% QF-1 at 185 "C and the corresponding derivatives of amino sugars on 2.2% SE-30 at 205 "C. Monomethylated hexoses were determined on 1 % OV-225 at 200 "C. Detection was carried out by total ionization current and mass fragmentography at m/e values 129, 161, 189 and 233 for neutral sugars and 158 for amino sugars. In the identification of (2-0)-substituted galactose, (6-0)-substituted mannose and (4-0)-substituted glucose it was necessary to use NaB2H, instead of NaBH, in the reduction of the partially methylated sugars and detection was carried out at mje values 189 and 190. The m/e values used in the present study were selected from reported spectra of partially methylated alditol acetates of hexoses [14- 161 and hexosamines [13,17,18] in order to make possible the selective detection of differentially substituted sugars.

Retention times of partially methylated alditol acetates were compared with those obtained from reference oligosaccharides and glycopeptides and with values reported previously [16,18,19]. Response factors for various differentially substituted neutral sugars in mass fragmentographic detection at different mje values were obtained from the reference oligosaccharides and glycopeptides : N-acetylneuraminyl-(2-3)-lactose, N-acetylneuraminyl-(2-6)-lactose, lacto-N-fucopentaose I, transferrin and fetuin N-glycosidic glycopeptides. Since no reference compound containing (2,6-di-O)-substituted mannose and (3,4,6-tri-O)-substituted mannose was available, the response factors of the corresponding methylated hexitols were calculated on the basis of the reported mass spectra [16]. The relative amounts of the partially methylated hexosamines were calculated from the peak areas obtained by monitoring of the ion mje 158, the intensity of which has been shown to be the same in the spectra of all partially methylated alditol acetates of hexosamines [18].

The methyl substitution patterns were confirmed by mass spectral analysis [13 - 181. Mass spectra were recorded with a Varian MAT CH-7 mass spectro-

meter equipped with SpectroSystem 100 MS data- processing system. The ionization potential was 70 eV and ionization current 300 PA. An Altema AL 5 multiple ion detector was used for mass fragmentographic detection.

Materials

Materials were obtained as follows : Sephadex G-1 0, G-25, G-50 and concanavalin AlSepharose, Pharmacia Fine Chemicals, Sweden ; NaB'H,, Merck. Vibrio cholerae neuraminidase (grade B) was obtained from Calbiochem and a-L-fucosidase from beef kidney was purchased from Boehringer Mannheim. A mix- ture of N-acetylneuraminyl-(2-3)-lactose and N-acetylneuraminyl-(2-6)-lactose, fetuin (type II), transferrin (grade 11) and a-methyl-D-glucoside were obtained from Sigma Chemical Company. Glycopeptides from transferrin and fetuin were prepared and fractionated as described for glycopeptides from tissues. N-Acetyl- neuraminyl-(2-6)-lactose and lacto-N-fucopentaose I were kindly supplied by Dr A. Gauhe (Heidelberg).

RESULTS

Carbohydrate Composition of Purijied Glycoproteins

The amounts of dry lipid-free residue obtained from 1 g of tissue (fresh weight) were: brain, 100 mg; kidney 160 mg and liver 230 mg. The amount of total glycopeptide carbohydrate per lipid-free residue varied considerably in the tissues (Table 1) being highest in kidney and lowest in liver. The high amount of lipid- free residue in liver and its low content of glycopeptide carbohydrate suggest that liver tissue contains proportionally less glycoproteins compared to other proteins than brain and kidney.

The carbohydrate composition of the total purified glycopeptides was similar in different tissues (Table 1). The most prominent difference was the high proportion of fucose in brain glycopeptides.

Fractionation of Gly copeptides

Glycopeptides were fractionated by affinity chromatography on concanavalin AjSepharose into three fractions (Fig. 1). Concanavalin A is known to bind only glycopeptides, which contain at least two terminal unsubstituted or (2-0)-substituted a-mannose residues [20,21]. Therefore, acidic N-glycosidic glycopeptides with two peripheral branches such as that of transferrin are weakly bound to the lectin and neutral mannose-rich N-glycosidic glycopeptides containing several terminal and (2-0)-substituted a-mannose residues are strongly bound to the lectin [21]. The rest


Table 1. Carbohydrate composition of purified glycopeptides from different rat tissues. Values are given as pmol sugar/100 mg lipid-free residue

Carbohydrate Amount present in

brain kidney liver

pmo1/100 mg mol/l00 mol Fucose 0.645 9.2 Mannose 2.17 31 .O Galactose 1.22 17.5 GalNAc 0.320 4.6 GlcNAc 1.65 23.6 AcNeu 0.980 14.0 Total 6.98 100

pmo1/100 mg mo1/100 mol 0.457 5.4 2.36 28.2 1.85 22.1 0.301 3.6 2.37 28.4 1.03 12.3 8.37 100

pmo1/100 mg mo1/100 mol 0.055 2.2 0.928 36.4 0.470 18.5 0.072 2.8 0.637 24.9 0.385 15.1 2.55 100

Total purified glycopeptides

affinity chromatography on Concanavalin AlSepharose

not bound 20mM aMeGlc 200mM aMeGlc

NaOHINaBH, Sephadex (3-50 I

A I I 0-glycosidic fraction A fraction B friction c oligosaccharides Fig. 1. Scheme for the frationation oftissue glycopeptides. aMeGlc, a-methy1-D-glucoside

0.3 BD

0.2 - 4 A

0.1 -

5 0 I

0

* 0.2

0 B c m 0.1

n

-

a . I

of the glycopeptides do not interact with concanavalin A and are eluted with the starting buffer.

Glycopeptides not bound to concanavalin A were treated with NaOH/NaBH, and subjected to gel filtration on Sephadex G-50 (Fig. 2). Two carbohydrate fractions were obtained. The fraction eluted first (fraction A) contained N-acetyl-glucosamine, mannose, galactose, fucose and N-acetylneuraminic acid and thus represented alkali-stable N-glycosidic glycopeptides. The second fraction, which was mainly composed of N-acetylgalactosaminitol, galactose and N-acetylneuraminic acid, contained the released 0- glycosidically-linked oligosaccharides. Small amounts of mannose and N-acetylglucosamine were also present in this fraction probably due to tailing of the peak of the N-glycosidic glycopeptides. The amounts of N- acetylgalactosaminitol and galactose correlated with that of ~-galactosyl-(l-3)-N-acetylgalactosaminitol, which indicates that the 0-glycosidically-linked oligosaccharides from kidney and liver are mainly composed of ~-galactosyl-(l-3)-N-actylgalactosamine and its monosialosyl and disialosyl derivatives as is the case in brain [7]. The amounts of these compounds in the three tissues have been reported previously [22].

Since no N-acetylgalactosaminitol was found in the N-glycosidic glycopeptide peak eluted first, no large molecular size alkali-labile O-glycosidically- linked carbohydrate chains such as those found in the mucus of the digestive tract and ovarian cysts [23,24] are present in these tissues.

0.2

0.1

‘15 2 0 25 30 35 40 45

Fig. 2. Separation of 0-glycosidic alkali-labile carbohydrate chains from N-glycosidic glycopeptides. Glycopeptides not bound by Con- canavalin A were treated with NaOH/NaBH,, desalted on Sepha- dex G-10 (3 cm x 70 cm) and subjected to gel filtration on Sephadex G-50 (2 cm x 75 cm). The column was eluted with 0.1 M pyridine/ acetic acid buffer (pH 5.0). Fractions of 5 ml were collected and analyzed for neuraminic acid. For comparison the elution volumes of blue dextran (BD) and ~-galactosyl-(l-3)-N-acetylgalactosamini- to1 (GG) are shown. (A) Brain glycopeptides; (B) kidney glycopeptides and (C) liver glycopeptides

Fraction number

Fraction B glycopeptides were composed of the same sugars as fraction A glycopeptides but the relative amounts of galactose and N-acetylneuraminic acid were lower than in fraction A. Glycopeptides containing only mannose and N-acetylglucosamine were found in fraction C. The carbohydrate composition of the three N-glycosidic glycopeptide fractions from kidney and liver were similar to the carbohydrate composition of brain glycopeptide fractions reported before [ 5 ] . However, the fractions showed small differences, which were similar to those pre- sented above (Table 1) for the unfractionated glycopeptides. The relative amount of fucose in both


Brain Kidney Liver

60 -

0"

4

20

10 h 1 - 1 O A B C O A B C O A B C

Fig. 3. Relative amounts of 0-glycosidic carbohydrate and different types of N-glycosidic glycopeptides in rat tissues. Glycopeptides were fractionated by Concanavalin-A-affinity chromatography and 0-glycosidic oligosaccharides were separated after NaOH/ NaBH, treatment by gel filtration on Sephadex G-50. The amount of 0-glycosidic carbohydrate in different tissues was taken from previous results [22]. Values are expressed as percentages of the sum of carbohydrate in all types of glycopeptides. (0) O-glycosidically-linked oligosaccharides; (A) fraction A glycopeptides ; (B) fraction B glycopeptides and (C) fraction C glycopeptides.

fraction A and B glycopeptides from brain was considerably higher than in kidney and liver glycopeptides.

The relative amounts of 0-glycosidically-linked oligosaccharides and different types of N-glycosidic glycopeptides in brain, kidney and liver are shown in Fig. 3. Kidney glycoproteins are characterized by a relatively low amount of 0-glycosidically-linked oligosaccharides and fraction C glycopeptides, whereas both these fractions are enriched in brain. Liver glycoproteins are characterized by a low amount of O-glycosidically-linked oligosaccharides and a high amount of fraction B glycopeptides.

Methylation Analysis

In order to obtain more detailed information about the structures of the carbohydrate chains, the substitution of the sugar residues of fraction A, B and C glycopeptides was studied by methylation analysis. The partially methylated alditol acetates were analyzed by gas-liquid chromatography and mass spectrometry. Since several partially methylated hexitols have nearly identical retention times on gas-liquid chromatography detection was performed by mass fragmentography. By selecting specific mje values for detection, differentially substituted hexoses with nearly identical retention times were identified and quantified separately (Fig. 4). In addition, because impurities originating from the biological samples and the methylation reagents interfere less in mass fragmento-

a- c 0 a

z L 0 c 0

a- c a- n

m / e 161 m / e 189 m / e 158

10

A

b 0 2 4 6 8 0 2 4 6 8 1 0 1 2 0 2 4 6 8

5 m/e158 I m / e 161 m / e 189 B

I , , , , 4 3 I I I " I I I I I

0 2 4 6 8 0 2 4 6 8 1 0 1 2 0 2 4 6 8

2

0 2 4 6 8 0 2 4 6 8 1 0 1 2 0 2 4 6 8

Fig. 4. Mass fragmentograms of partially methylated alditol acetates obtained from rat brain glycopeptides. (A) Fraction A glycopeptides; (B) fraction B glycopeptides and (C) fraction C glycopeptides. Peak 1, 2,3,4-tri-O-methylfucitol; peak 2, 2,3,4,6-tetra-O-methylmannitol and 2,3,4,6-tetra-0-methylglucitol; peak 3, 2,3,4,6-tetra- 0-methylgalactitol; peak 4, 2,4,6-tri-O-methylgalactitol; peak 5, 3,4,6-tri-O-methylmannitol; peak 6, 2,3,4-tri-O-methylmannitol; peak 7, 2,3,4-tri-O-methylgaIactitol; peak 8, 3,6-di-O-methylmannitol; peak 9, 2,4-di-O-methylmannitol; peak 10, 3,4-di-0- methylmannitol; peak 11, 2-deoxy-2-N-methylacetamido-3,4,6-tri- 0-rnethylglucitol; peak 12, 2-deoxy-2-N-methylacetamido-3,6-di- 0-methylglucitol ; peak 13, 2-deoxy-2-N-methylacetamido-6-mono- 0-methylglucitol; peak 14, 2-deoxy-2-N-methylacetamido-3-mono- 0-methylglucitol. Abscissa: retention time in minutes. Conditions: 3% QF-1, 185 "C for neutral sugars and detection by mass fragmentography at m/e values 161 and 189. 2.2% SE-30, 205 "C for amino sugars and detection by mass fragmentography at m/e 158

Retention time

graphic detection, accurate results were obtained even from samples containing a few nmoles of each sugar (about 10 pg of total carbohydrate).

Fraction A Glycopeptides

In all tissues studied the methylation products of mannose in fraction A glycopeptides were derived


Table 2. Relative amounts of partially methylated alditol acetates of neutral sugars in different N-glycosidic glycopeptides derived from brain, kidney and liver. Values are expressed as moles per three moles of mannose. A, fraction A glycopeptides; B, fraction B glycopeptides and C, fraction C glycopeptides

Component Glc peak Glycosidic Brain Kidney Liver number linkage

A B C A B C A B C

2,3,4-Tri-O-methylfucitol 1

2,4,6-Tri-O-methylgalactitol 4 2,3,4-Tri-O-methylgalactitol 7

3,4,6-Tri-O-methylmannitol 5

2,4-Di-O-methylmannitol 9 3,4-Di-O-methylmannitol 10

2,3,4,6-Tetra-O-methylgalactitol 3 3,4,6-Tri-O-methylgalactitol ~

2,4-Di-O-methylgalactitol -

2,3,4-Tri-O-methylmannitol 6 3,6-Di-O-methylmannitol 8

2-Mono-O-methylmannitol ~

terminal terminal 2 3 6 3 and 6 2 6 2 and 4 3 and 6 2 and 6 3,4 and 6

1.5 1.2 0.0" 0.6 0.6 0.0 0.4 0.2 0.0 1.6 0.8 0.0 1.4 0.7 0.0 0.5 0.2 0.0 0.0 0.0 0.0 n.b.b n.d. 0.0 n.d. n.d. 0.0 2.6 0.8 0.0 2.5 0.9 0.0 2.4 0.5 0.0 0.2 0.2 0.0 0.7 0.6 0.0 1.1 1.1 0.0 0.1 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.7 1.9 1.0 0.8 2.1 1.0 1.0 2.0 1.2 0.0 0.0 0.5 n.d. n.d. 0.8 n.d. n.d. 0.7 0.7 0.0 0.0 0.7 0.0 0.0 0.6 0.0 0.0 0.6 0.4 1.5 0.4 0.3 1.1 0.9 0.8 1.1 0.7 0.3 0.0 0.7 0.3 0.0 0.4 0.2 0.0 0.3 0.4 0.0 0.4 0.3 0.0 0.1 0.0 0.0

a O.O=less than 0.05, n.d. =not determined, ' absent in Fig. 4

from (2-0)-substituted, (2,4-di-O)-substituted, (2,6- di-0)-substituted, (3,6-di-O)-substituted and (3,4,6- tri-0)-substituted mannose (Table 2). These methylation products except that obtained from (3,4,6-tri-0)- substituted mannose are the same as those from the N-glycosidically-linked oligosaccharide of fetuin. In contrast to the structure described previously for this soluble glycoprotein [25] fetuin was also found to contain trace amounts of (2,6-di-O)-substituted mannose. The N-glycosidically-linked oligosaccharide of fetuin, which does not bind concanavalin A, has been shown to be composed of a core pentasaccharide, mannotriosido-di-N-acetylchitobiose, to which three peripheral AcNeu-Gal-GlcNAc-branches are attached (Fig. 5). In some glycoproteins the proximal (3,6-di-0)- substituted mannose residue is also substituted at C-4 by N-acetylglucosamine [26,27] constituting (3,4,6- tri-0)-substituted mannose similar to that present in fraction A glycopeptides. Since the relative amounts of the methylation products of mannose in fraction A glycopeptides differ considerably from those expected for fetuin, probably heterogeneity is present at the mannose branchpoints. Furthermore, since the ratio of the sum of the methylation products of mannose to that of galactose was about 3:4 (Table 2), fraction A glycopeptides probably contain on average about four peripheral branches.

The ratios of the methylation products of mannose probably derived from the core region of fraction A glycopeptides varied little in the three tissues. In contrast to this, there were tissue specific differences in the substitution pattern of galactose and N-acetylglucosamine, which together with N-acetylneuraminic acid and fucose form the peripheral branches in known soluble glycoproteins (Fig. 5) . Brain glycopeptides contained mainly (3-0)-substituted galactose

(Table 2). Small amounts of (6-0)-substituted and traces of (2-0)-substituted and (3,6-di-O)-substituted galactose were also found. More than 85% of the substituents of galactose at C-3 and C-6 had disappeared after neuraminidase digestion, which indicates that galactose is substituted by N-acetylneuraminic acid. The presence of considerable amounts of terminal non-substituted galactose and glucosamine suggests that the peripheral branches in brain glycoproteins often are incomplete. Kidney glycopeptides are characterized by a higher amount of (6-0)-substituted galactose and (3,6-di-O)-substituted galactose than brain glycopeptides. The amount of terminal unsubstituted glucosamine was also low. Liver glycopeptides differed from brain and kidney glycopeptides in the amount of terminal unsubstituted sugar residues. Liver glycopeptides contained no terminal glucosamine and the amount of terminal galactose was also low, which suggests that the peripheral branches are mainly complete. Furthermore, liver glycopeptides contained the highest proportion of (6-0)-substituted galactose residues.

In all tissues studied the major methylation product of N-acetylglucosamine found was derived from (4-0)-substituted glucosamine (Table 3). In brain, fraction A glycopeptides contained considerable amounts of (3,4-di-O)-substituted and (4,6-di-O)-substituted glucosamine, whereas in kidney and liver the amounts of these (di-0)-substituted glucosamine derivatives were low. The methylation product derived from (4,6-di-O)-substituted glucosamine is expected from several glycoproteins containing fucose bound at C-6 of the proximal N-acetylglucosamine residue of the carbohydrate chain [26-291. In brain glycopeptides the two (di-0)-substituted glucosamine derivatives were unaffected by neuraminidase digestion.


Table 3. Relative amounts of partially methylated alditol acetates of amino sugars in different N-glycosidic glycopeptides derived from brain, kidney and liver Values are expressed as moles per mole of 2 deoxy-2-N-methylacetamido-3,6-di-O-methylglucito1. A, fraction A glycopeptides; B, fraction B glycopeptides and C, fraction C glycopeptides

2-Deoxy-2-N-methylacetamido- Glc peak Glycosidic Brain Kidney Liver number linkage

A B C A B C A B C

-3,4,6-tri-O-methylglucitol 11 terminal 0.2 0.8 0.1 0.1 0.2 0.1 O.Oa 0.0 0.0 -3,6-di-O-methylglucitol 12 4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 -6-mono-0-methylglucitol 13 3 and 4 0.3 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -3-mono-0-methylglucitol 14 4 and 6 0.1 0.1 0.0 0.1 0.1 0.0 0.0 0.0 0.0

a 0.0 =less than 0.05

However, more than 60% of (3,4-di-O)-substituted and about 80 % of the (4,6-di-O)-substituted glucosamine had disappeared after fucosidase digestion and the relative amount of (4-0)-substituted glucosamine was increased. Furthermore, both the (di-0)-substituted glucosamine derivatives disappeared quantitatively by treatment with 0.1 M HCl at 100 "C for 1 h.

These results indicate that the substituents of glucosamine at C-3 and C-6 are fucose residues, which also correlates with the amount of fucose in different tissues (Tables 1 and 3). Since the amounts of other methylation products were unaffected in these experiments and only traces of (2-0)-substituted galactose were found in brain glycopeptides, it may be concluded that fucose is mainly attached to N-acetylglucosamine. The presence of glucosamine substituted at C-3 by fucose in N-glycosidic carbohydrate chains has not previously been reported.

Fraction B Glycopeptides

In contrast to fraction A glycopeptides, only traces of (2,4-di-O)-substituted mannose were present in fraction B glycopeptides (Table 2). The ratio of (2-0)- substituted mannose to the sum of (2,6-di-O)-substituted, (3,6-di-O)-substituted and (3,4,6-tri-O)-substituted mannose was about 2: 1 in all tissues studied. Furthermore, the ratio of the sum of the methylation products of mannose to that of galactose was about 3 : 2. These results suggest that these glycopeptides contain a similar structure as that described for transferrin and the carbohydrate chains of immunoglobulins [27,30] (Fig. 5). Transferrin, which is also bound to concanavalin A, contains the same core pentasaccharide as fetuin but with only two peripheral AcNeu-Gal-GlcNAc-branches. However, whereas the branching point mannose of transferrin is (3,6-di-0)- substituted, in tissue glycoproteins it is either (3,6-di- 0)- or (2,6-di-O)-substituted. In addition, part of the (3,6-di-O)-substituted mannose residues also are substituted at C-4 as in immunoglobulins [26,27].

The substitution pattern of galactose and N-acetylglucosamine in fraction B glycopeptides from different tissues was similar to that of fraction A glycopeptides. However, on average fraction B glycopeptides contained proportionally more (6-0)-substituted galactose, terminal galactose and terminal glucosamine than fraction A glycopeptides (Tables 2 and 3). This clearly indicates that fraction B glycopeptides differ from fraction A glycopeptides not only in the structure of the core region but also in the size and substitution pattern of their branches. We have also shown that the relative amount of disialosyl groups is considerably higher in fraction A glycopeptides than in B glycopeptides [31].

Fraction C Glycopeptides

The methylation products of mannose obtained from fraction C glycopeptides were derived from (2-0)-substituted, (6-0)-substituted, and (3,6-di-0)- substituted mannose (Table 2). Only traces of (2,4-di- 0)-substituted and (2,6-di-O)-substituted mannose were present. Terminal unsubstituted mannose was not quantitated because of the presence of a-methyl-D- glucoside originating from the elution buffer used in concanavalin-A-affinity chromatography. a-Methyl- D-glucoside gives a methylation product with a nearly identical retention time and mass spectrum as terminal mannose. Based on the results of the methylation analysis the branchpoints of fraction C glycopeptides are composed of (3,6-di-O)-substituted mannose residues and the mannose residues in the chains are bound by 1 - 2 and 1 - 6 linkages to each other. Brain glycopeptides contain proportionally more branch-point mannose residues to mono-substituted mannose residues than kidney and liver glycopeptides. In addition to the mannose residues also (4-0)-substituted and small amounts of terminal unsubstituted glucosamine were present. These results suggest that fraction C glycopeptides from these tissues resemble by their structure mannose-rich glycopeptides from ovalbumin (Fig. 5) [32] and thyroglobulin [33].


Table 4. Relative amounts of differentially substituted galactose residues in soluble and membrane-bound glycoproteins derived from rat brain and liver

Source Unsubstituted (3-O)-substituted (6-O)-substituted Total

Liver Microsomal fraction Soluble fraction

Microsomal fraction Soluble fraction

Brain

10.6 8.7

44.2 31.4

65.3 46.7

49.2 51.8

24.1 44.6

6.6 16.8

100 100

100 100

Methylation Analysis of Glycopeptides Obtained from Microsomal and Soluble Fractions

Purified glycopeptides from microsomal and soluble fractions were subjected to methylation analysis. Similar differences in the substitution pattern of galactose and N-acetylglucosamine were found as described above for whole tissue. The only prominent difference was the higher proportion of (6-O)-substituted galactose in the soluble glycoproteins of both tissues (Table 4). The results suggest that the carbohydrate chains of both soluble and membrane-bound glycoproteins derived from brain are characterized on average by incomplete peripheral branches and the substitution of galactose at C-3, whereas complete peripheral branches and the substitution of galactose both at C-3 and C-6 are typical of soluble and membrane-bound glycoproteins derived from liver. This indicates that the glycosylation pattern of the peripheral branches of both soluble and membrane-bound glycoproteins is characteristic to each tissue, and the differences observed for glycopeptides derived from whole tissue are not due to differences in the proportions of membranes in the tissues.

DISCUSSION

Tissue glycoproteins from brain, kidney and liver of rat contain four major structurally distinct types of oligosaccharides : 0-glycosidically-linked oligosaccharides, two different types of acidic N-glycosidically-linked oligosaccharides and mannose-rich N-glycosidically-linked oligosaccharides. The carbohydrate composition and the methylation products obtained from the oligosaccharides of the tissue glycoproteins suggest that the average structures of these oligosaccharides resemble those of the soluble glycoproteins.

The 0-glycosidically-linked oligosaccharides from kidney and liver were mainly b-galactosyl-( 1 -3)-N- acetylgalactosamine and its monosialosyl and disialosyl derivatives as is the case in brain [7]. The same disaccharide and its sialosylated derivatives have been shown also to occur in other tissues [22] and in

erythrocyte, lymphocyte and milk fat globule membranes [34 - 361. We have previously reported that both the total amount of 0-glycosidically-linked oligosaccharides and the relative proportions of the different 0-glycosidically-lin ked oligosaccharides vary from one tissue to another [22]. Furthermore, brain glycoproteins contained an 0-glycosidically-linked oligosaccharide, cc-galactosyl-(l-3)-N-acetylgalactosamine, which was not found in other tissues [22]. The results of the present study suggest that kidney and liver do not contain large molecular size O-glycosidically-linked carbohydrate chains similar to those present in the mucus of the digestive tract and in ovarian cyst fluids [23,24].

Both acidic N-glycosidic glycopeptides of fraction A and B in all tissues studied seem to have a more variable structure than the N-glycosidic carbohydrate chains of fetuin and transferrin, which behave similarly on concanavalin-A-affinity chromatography, respectively. Acidic N-glycosidic carbohydrate chains from tissue glycoproteins contain, in contrast to the reference glycoproteins, considerable amounts of (2,6-di-O)-substitutedmannose. Thecarcinoembryonic antigen and the oligosaccharides present in tissues of patients with fucosidosis have also been shown to contain significant amounts of (2,6-di-O)-substituted mannose [37,38]. The structure of the acidic N-glycosidic carbohydrate chain of the carcinoembryonic antigen is unknown, whereas an average structure similar to that of transferrin (Fig. 5) has been proposed for the oligosaccharides present in fucosidosis [38]. The hepatic glycoprotein responsible for the uptake of asialoglycoproteins from circulation has been proposed to contain a core structure composed of only two mannose residues and the chitobiose unit to which three peripheral AcNeu-Gal-GlcNAc-branches are attached [39]. This suggests that the core structure of this specific hepatic glycoprotein differs from known soluble glycoproteins [25 - 301 and probably from the average structure of liver glycoproteins.

The peripheral branches of known soluble glycoproteins are usually composed of N-acetylneuraminic acid, galactose and N-acetylglucosamine (Fig. 5). Usually N-acetylneuraminic acid is bound by 2 - 3 and/or 2-6 linkages to galactose, which is attached


AcNeu AcNeu AcNeu

Gal Gal Gal

GlcNAc GlcNAc GlcNAc

I a2-6(3) I a2-3(6) I a2-3(6)

I 01-4 I 81-4 I 81-4 AcNeu I 012-3

Gal ot2-6 I 81-3

AcNeu - GalNAc

l a Ser

0-glycosidically -linked oligosaccharide from rat brain I 81-4

I 8

GlcNAc I 81-4

GlcNAc

Asn

AcNeu AcNeu I a2-6(3) I a2-3(6)

Gal Gal I 01-4 1 81-4 GlcNAc GlcNAc I 01-2 I 81-2

/Man a l -6

Man

GlcNAc

GlcNAc

Asn

N- Glycosidically -linked oligosaccharide from transferrin

1 81-4

I 01-4

1 0

N-Glycosidically -linked oligosaccharide from fetuin

Man Man Man \ \ /

a1 -2 \ 011-3 /a1-6 Man ‘Man \ /

011-3, ,(~1-6

Man I 01-4 GlcNAc I 81-4

GlcNAc

Asn I 0

N-Glycosidically -linked oligosaccharide V from ovalbumin

Fig. 5 . Proposed structures of different types of carbohydrate chains of glycoproteins [7,25,30,32]

to the C-4 of N-acetylglucosamine. It has also been reported that the galactose may be substituted at C-2 by a fucose residue [38]. The peripheral branches are not always complete. Immunoglobin G contains peripheral branches composed of only N-acetylglucosamine and galactosyl-N-acetylglucosamine [40]. After mild acid hydrolysis of brain, kidney and liver glycopeptides, P-galactosyl-( l -4)-N-acetylglucosamine was detected by gas-liquid chromatography as the major component in the N-acetylhexosamine-containing disaccharide fraction (Krusius, T. and Finne, J., un- published results), which indicates that the peripheral branches of tissue glycoproteins are similar to those of soluble glycoproteins. However, the results of the methylation analysis in the present study suggest that the completeness of the peripheral branches varies in different tissues. Brain glycoproteins are characterized by relatively incomplete branches as indicated by the proportionally high amount of terminal unsubstituted galactose and N-acetylglucosamine. In liver glycopro-

teins the peripheral branches seem to be mainly complete. Furthermore, variations in the site of attachment of N-acetylneuraminic acid to galactose were observed from one tissue to another. N-Acetylneuraminic acid is mainly bound to C-3 of the galactose in brain glycoproteins. In liver, galactose is substituted nearly as often at C-6 as at C-3. These differences occur both in the soluble and membrane-bound glycoproteins of tissues. Although this suggests similarity in the bio- synthesis of at least the peripheral parts of soluble and membrane-bound glycoproteins, we have, on the other hand, previously found that disialosyl (a - N - acetylneuraminyl - (2 - 8) - N - acetylneuraminyl) groups are practically only present in membrane- bound glycoproteins [41].

Brain glycoproteins contain proportionally about 2 and 4 times more fucose than liver and kidney glycoproteins, respectively. Based on the results of fucosidase and mild acid treatment coupled to methylation analysis fucose was found to be bound mainly to


N-acetylglucosamine at C-3 and C-6 and not to galactose. This is in agreement with the results of Yamashita et al. who have proposed that fucose is attached in type 2 ABO-blood-group-active carbohydrate chains (Gal-(l4)-GlcNAc.. .) to C-3 of the N-acetylglucosamine, whereas in type 1 chains (Gal- (1-3)-GlcNAc. . .) fucose is bound to C-2 of the galactose [42]. N-Glycosidic carbohydrate chains of several known soluble glycoproteins contain fucose attached to C-6 of the proximal N-acetylglucosamine residue [26-291, whereas fucose is bound to C-2 of the galactose in oligosaccharides (probably derived from N-glycosidic carbohydrate chains) of patients with fucosidosis [ 3 81. N-Gl ycosidic carbohydrate chains have previously not been shown to contain fucose at C-3 of N-acetylglucosamine as found in the present study. Since by methylation analysis no information is obtained about the sequence of the sugar residues, it can not be concluded whether fucose is attached to the C-3 of the N-acetylglucosamine residues in the peripheral branches or in the core region. If the N-acetylglucosamine residues located in the peripheral branches are substituted at C-3 by fucose, a sugar sequence similar to that of the X-antigen present in glycolipids [43 - 451 also would occur in the N-glycosidic carbohydrate chains of brain glycoproteins.

The present study shows that although the substitution pattern of the mannose residues probably derived from the core region of the carbohydrate chains are similar to each other, considerable differences occur in the peripheral parts of the carbohydrate chains in different tissues. Terminal sugar sequences have been shown to be the basis for several biological phenomena such as the specificity of ABO-blood groups [46] and the interactions of thyrotropin, human chorionic gonadotropin and cholera toxin with their receptors [47 - 491. Since the carbohydrate chains of cell surface glycoproteins are proposed to take part in interactions between the cell and its environment, for instance cell recognition and adhesion, the tissue differences in the terminal parts of the carbohydrate chains observed in the present study may be important for the specificity of these phenomena.

The skilful technical assistance of Mrs Maire Ojala, Mrs Liisa Kuivalainen, Mrs Hilkka Ronkko and Mrs Kristiina Heinonen is appreciated. This work was supported by the Sigrid Jusklius Foundation.

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T. Krusius and J. Finne, Laaketieteellisen kemian laitos, Helsingin Yliopisto, Siltavuorenpenger 10A, SF-00170 Helsinki 17, Finland

Documents

Structural Features of Tissue Glycoproteins. Fractionation and Methylation Analysis of Glycopeptides Derived from Rat Brain, Kidney and Liver