High-performance liquid chromatographic analysis of oligosaccharides and glycopeptides accumulating in lysosomal storage disorders

ANALYTICAL

BIOCHEMISTRY

102,

213-219

(1980)

High-Performance Liquid Chromatographic Analysis of Oligosaccharides and Glycopeptides Accumulating in Lysosomal Storage Disorders N. M. K. NG YING Depurtment

of Neurochemistry. 3801

University

Montreal Street,

KIN

AND L. S. WOLFE

Neurologicul Institute and Hospital, Montreal, Quebec H3A 2B4. Canudo

Received

July

McGill

Unir2ersity.

11. 1979

A high-performance liquid chromatography method was developed to separate the oligosaccharides and glycopeptides which accumulate in the liver and are excreted in the urine of a number of lysosomal storage disorders. Characteristic elution profiles were obtained for oligosaccharides, their borohydride reduced products, and glycopeptides in mannosidosis. fucosidosis, G,,,,-gangliosidosis, G,,-gangliosidosis variant 0, sialidosis, and aspartylglucosaminuria. Chromatographic separations could be completed in 20 min.

In patients with autosomal recessive inherited lysosomal storage disorders, such as G,,-gangliosidosis Types 1 and 2, GMzgangliosidosis, variant 0 (Sandhoff-Jatzkewitz disease), mannosidosis, fucosidosis, aspartylglucosaminuria, and sialidosis, the absence of specific glycosylhydrolases results in an incomplete catabolism of various cellular and plasma glycoproteins. Thus, characteristic oligosaccharides and glycopeptides are stored in tissues and excreted in the urine of these patients (l- 16). In these disorders, typical profiles of urinary oligosaccharides can be demonstrated by thinlayer chromatography on silica gel (17). For structural studies the oligosaccharides and glycopeptides from these patients have been fractionated and isolated using Bio-Gel P chromatography in our laboratory (18). The method is, however, time consuming. Recently, we investigated the possibility of using high-performance liquid chromatography (hplc)’ for the rapid fractionation of these oligosaccharides and glycopeptides. High-pressure liquid chromatography has been successfully used in the analysis of ’ Abbreviations used: hplc, high-pressure liquid chromatography: Man. mannose: GlcNAc, N-acetylglucosamine; Fuc, fucose: NeuNAC. N-acetylneuraminic acid: Gal. galactose; GalNAc. galactosamine. 213

cereal hydrolysates (19,20), enzymic hydrolysates of chondroitin sulfates (21), and oligomers of N-acetylglucosamine (22). MATERIALS

AND METHODS

Sample preparation. Oligosaccharides and glycopeptides of known structures (see Table 1) were isolated from pathological urine and tissue samples by Bio-Gel P-6 and P-2 chromatography as described earlier (1,18). Reduction of oligosaccharides was carried out at room temperature for 4 h using an equal weight of sodium borohydride. Excess borohydride was destroyed by addition of acetic acid and the sample degassed before hplc analysis. For hplc analysis of urine, samples were prepared in the following manner: aliquots (5 ml) from a 24-h urine collection were passed through a Sep-Pak CIB cartridge (Waters Associates, Mississauga, Ontario) which resulted in almost complete decolorization, and then consecutively through two small columns of Dowex 5OW-X8 (H+) and Dowex l-X8 acetate resins. The desalted samples were then evaporated to dryness in t’acuo and dissolved in 200 ~1 of water. Ten to twenty-five microliters of the various samples was required for each hplc run. 0003-2697/80/030213-07$02,00/O Copyright % 1980 by Academic Press, Inc. All rights of reproductmn in any form reerved.

214

KIN

AND

WOLFE

TABLE STRUCTURES URINE

OF OLIGOSACCHAR~DES

IN DIFFERENT

AND

LYSOSOMAL

I

GLYCOPEPTIDES

STORAGE

ACCUMULATING

DISEASES

AFFECTING

IN TISSUES

AND

THE CATABOLISM

EXCRETED

IN THE

OF GLYCOPROTEINS

Compound

NO

Disease

StrUCtWe

References

-

1

LI Man I-3 p Man

2

/3 GlcNAc

3

/3 Gal I-4 p GlcNAc

4

p GIcNAc

l-2

a M;n

p GlcNAc

l-2

a Man l-6’

i-U4

I-4 GIcNAc(~,P)

u Man I-3 /3 Man l-4

Mannosidosis

(7-9)

G,,-Gangliosidosis. variant 0

(5.6)

G,,-Gangliosidosis. Type l

(3.11)

G.,-Gangliosidosis, variant 0

(4)

G,,-Gangliosidosis, variant 0

14)

G,,-Gangliosidosis, variant 0

(6)

GlcNAcla.P)

G.,-Gangliosidosis, TYPE l

(1,2)

fo one of these positions

G,,-Gangliosidosls. Type l

(I .2)

Fucosidosis

(IO)

Fucosidosis

(l%Il)

Fucosidosis

(II)

Aspartylglucos. aminuria

(12.13)

GlcNAc(cx.~)

I-2 a Man I-316 p Man I-4 GlcNAcla./3)

R’ l-3 ‘0

Man I-4

A,

R’

5

as in 4 with R’ = /3 GlcNAc

6

as in 4 with R’ = H. R2 = p GlcNAc

1

PGal

I-4fiGlcNAc

GlcNAcloJ3)

I-2a

linked

PI-4

IO one of these positions.

hnked

R2 = H

PI-4

P Man l-3,

~GalI-4/3GlcNAcI-2aMpn

l-6

if3

Man l-4

R R=H 8

as in 7 with R = p Gal l-4 GlcNAc

9

OLFuc I-6p

10

p Gal I-4 @GlcNAc a Fuc l-3

11

GlcNAc

linked

PI-4

I-Asn I-214

a Man I-316p

I-2/4a

Man I-316p

Man l-4

GlcNAcla,P)

/

/3Gal I-4PG)cNAc 01 Fuc l-3

Man I-4/3GlcNAc

I-4p

GlcE;Ac

I-Asn

a Fuc I-316

12

/3 GlcNAc

I-Asn

13

m NeuNAc

2-316 fl GalNAc

14

a NeuNAc

2-316 @ Gal I-4 p GlcNAc

15

Structure 7 with two additional ferminal /3 Gal

l-O&r l-214

o( NeuNAc

OLMan I-3/6p linked

2-3

Apparatus. All separations were made on a Waters Associates instrument with a Model M-6000A solvent delivery system, Model U6K manual injector, and Model R401 differential refractometer. The hplc was performed on a Waters Associates PBondapaklcarbohydrate column (30 cm x 4 mm i.d.). Chromatographic conditions. All operations were carried out at room temperature.

Man l-4

and/or

2-6

Sialidosis

(16)

GlcNAclu.~)

Sialidosis

(14.15)

to nonreducing

Sialidosis

(14-16)

The flow rate was 1.0 ml/min and the differential refractometer attenuation set at x4. The solvents used for isocratic elution were acetonitrile-1% acetic acid in water, 60:40 (v/v) for sialic acid-free oligosaccharides and glycopeptides, and acetonitrile0.1 M sodium acetate/acetic acid buffer, pH 5.6, 55:45 (v/v) for sialyl oligosaccharides and glycopeptides. Thin-layer chromatography was carried

OLIGOSACCHARIDES

AND GLYCOPEPTIDES

IN STORAGE

DISORDERS

out on Quantum silica gel plates (Pierce Chemicals, Rockford, Ill.). Plates were developed overnight in butan-I-ol-acetic acid-water, 2:1:1, and the compounds detected with the orcinol/sulfuric acid (17) or resorcinol reagents (23). RESULTS

Sialic Acid-Free

Oligosaccharides

In Table 1 the structures of the major oligosaccharides and glycopeptides stored in tissues and/or excreted in the urine of a number of lysosomal storage disorders with defective catabolism of glycoproteins are listed. Throughout the text, individual compounds are referred to by the number in the first column. Figures lA-C illustrate the elution profiles obtained for the linear tri-, tetra-, and pentasaccharides (structures 1, 2, and 3, Table 1) excreted by patients with mannosidosis, G,,-gangliosidosis, variant 0, and G,,-gangliosidosis, Type 1, respectively. In each case the oligosaccharide was partially resolved into two components. These were shown to be due to a mixture of (Y and p anomers by borohydride reduction. Thus, the reduced trisaccharide I, and pentasaccharide 3 each eluted as a single peak (Figs. 1D and F, respectively). In the case of the reduced tetrasaccharide 2, two exoisomers were separated which represent N-acetylglucosamine linked either to the C-2 or the C-4 position of the a-linked mannose (Fig. 1E). The major 2-linked isomer (5) eluted a little after the 4-linked isomer. It is of interest to note that the endoisomers of the pentasaccharide 3, i.e., 3-P-Man- and 6-P-Man-, were not separated. The oligosaccharides 4, 5, 7, and 8 (Table 1) with the commonbranched trimannosyl core found in many mammalian glycoproteins (24) also gave characteristic elution profiles in the hplc system. Thus the mixture of hexa- and heptasaccharides 4 and 5, respectively, stored in the liver of patients with GM2gangliosidosis, variant 0 (4), eluted at 5.5

FIG. 1. High-pressure liquid chromatography of the trisaccharide 1 (A), tetrasaccharide 2 (B), and pentasaccharide 3 (C), isolated from liver and/or urine of mannosidosis, GM,-ganghosidosis, and GM,-gangliosidosis patients, respectively (see Table 1 for structures). D-F show the corresponding borohydride-reduced compounds, 1 R, 2 R and, 3 R, respectively. All time scales are the same as in A. Detector response is the refractive index change at attenuation four times. The response shown in D represents 30 pg of carbohydrate injected. AI1 chromatographs are recoroed under exactly the same conditions.

min (Fig.2A) and the octasaccharide 7 and decasaccharide 8, present in G,,-gangliosidosis Type 1 patients (1,2), eluted at 7.3 min (Fig. 2B) and 9.3 min (Fig. 2C), respectively. Here again, as with the linear oligosaccharides described above, these branched chain oligosaccharides were partially separated into their a and p anomers. Thus, after borohydride reduction both the octa- and decasaccharides eluted as single peaks (Figs. 2E and F), whereas the mixture of hexaand heptasaccharides eluted as two distinct peaks (Fig. 2D, with the hexasaccharide as the major component eluting first. That the two peaks in Fig. 2B were due to the (Y and /3 anomers, respectively, of the octasaccharide 7 was further confirmed in the following manner. The compounds in

216

KIN AND WOLFE

FIG. 2. High-pressure liquid chromatography of the hexasaccharidel and heptasaccharide5 mixture isolated from the liver of a patient with GM,-gangliosidosis, variant 0 (A), the octasaccharide 7, and decasaccharided isolated from the liver and urine of patients with GM,-gangliosidosis, Type I (B and C). The corresponding borohydride-reduced compounds, 4 R, 5 R, 7 R, and 8 R are shown in D-F. Structures of the compounds are listed in Table 1. Other details are identical to Fig. 1.

each peak were isolated, the solvents removed, and the residue was equilibrated in water for half an hour before reinjecting into the hplc system. In each case, the isolated peak resolved again into the two anomers and gave exactly the same elution profiles as the original octasaccharide 7 in Fig. 2B. The hplc profiles obtained from concentrated and partially deionized urine from 24-h collections for several lysosomal storage disorders are shown in Figs. 3B-F. A representative normal urine profile prepared under the same conditions is shown in Fig. 3A. The compounds eluting in the first 3 min are the unresolved low molecular weight compounds present in normal urine. The (Y and p anomers of the trisaccharide I are the major components eluting after lactose (added as internal reference) in mannosidosis urine (Fig. 3B). The minor compounds eluting later represent oligosaccharides with a higher mannose content as reported in the literature (8,9). The profile for GM,-gangliosidosis urine (Fig. 3C) shows the elution of the major mixture of hexasaccharide 4 and heptasaccharides 5 and 6 together with smaller amounts of the tetrasaccharide 2. Under the conditions

used for elution the oligosaccharides 4, 5, and 6 were not completely resolved because of the (Y and p anomers. Reduction with sodium borohydride and altering the solvent elution system resolved these components. However, for rapid screening purposes the pattern shown is characteristic for this disease. The hplc elution profile for fucosidosis urine (Fig. 3D) is complex with three major components and several minor components which we have not chemically characterized as yet. The large peak eluting at 3.8 min is the small glycopeptide 9 described by several authors as the major urinary compound (10,25,26). CompoundlO eluting at 4.5-5.5 min is a mixture of the isomeric hexasaccharides with the characteristics 3 ,Clinked P-N-acetylglucosaminide recently described by Nishigaki et al. (10) and Ng Ying Kin and Wolfe (11). Compound 11 eluting at around 10.8 min is the larger glycopeptide recently described by us (11). The peak eluting at 13.5 min is an uncharacterized compound often seen in normal urine. GM,-Gangliosidosis urine also shows a very distinctive hplc elution profile (Fig. 3E). The major peak eluting at 7.3 min represents the a~3 anomers of the octasaccharide 7 (see also Fig. 2 for the profile

OLIGOSACCHARIDES

AND GLYCOPEPTIDES

IN STORAGE

DISORDERS

217

/ !a !I Monnosidosis GAQ-gongliosidosis 11

Normal

/

b-

3 min.

9

Fucosidosis F

E GM,-gongliosidosis

FIG. 3. The hole otofiles of mine samnles from lvsosomal storage disorders with defective catabolism of glycoproteins. Normal urine (A), mannosidosis (B), G,I-gangliosidosis, variant 0 (C), fucosidosis (D), GM,-gangliosidosis, Type 1 (E), aspartylglucosaminuria (F). The numbers over the peaks indicate the compounds listed with structures shown in Table 1. Each chromatogram was obtained under identical conditions. Other details are recorded in the legend to Fig. 1. The large detector response changes during the first 3 min of elution represent the low molecular weight materials present in urine.

of the isolated, purified compound). The structurally related decasaccharide 8 eluted at 9.3 min and a glycopeptide analog eluted at around 12.3 min (see 18). The mixture of the isomeric linear pentasaccharide 3 eluted much earlier at 4.3 min (see Fig. 1 for the hplc profile of the purified compound). The hplc profile for urine from an aspartylglucosaminuria patient (Fig. 3F) shows several well-separated sharp peaks reflecting the absence of a,/3 anomers associated with the reducing N-acetylglucosamine in the free oligosaccharides shown in Table 1. The major component is the glucosaminyl asparagine 22 eluting at 3.7 min. The compound

is followed by a series of glycosylasparagines with higher carbohydrate content as reported in the literature (12,13). Sialyl Oligosaccharides

The acidic solvent system used in the hplc of sialic acid-free oligosaccharides and glycopeptides would not elute sialic acid-containing oligosaccharides from the wB0ndapak.I carbohydrate column. Similarly with neutral acetonitrile-water systems the compounds were also strongly retained. A more ionic solvent system which contained sodium acetate in varying concentrations was found

218

KIN AND WOLFE Sialidosis

case much less decasaccharide 25 was present. The proportion of N-acetylneuraminic acid-linked a-2-3 and a-2-6 to galactosyl residues although markedly different in the two sialidosis phenotypes (16) were not reflected in the present hplc separation. Studies are in progress to achieve the separation of these isomers. DISCUSSION

4. The hplc profile of the urine from a patient with sialidosis, Type 2. The numbers refer to the structures listed in Table 1. NeuNAc is free N-acetylneuraminic acid. FIG.

to be satisfactory for hplc analyses of sialyl oligosaccharides. With a solvent system containing 45% 0.1 M sodium acetate-acetic acid buffer, pH 5.8, in acetonitrile, we were able to elute the major sialyl oligosaccharides excreted in the urine of patients with sialidosis. Typically, the hexasaccharide 14 is eluted at 5 min (Fig. 4). A smaller compound eluting earlier probably represents compoundl3, a small sialyl 0-glycosidically linked glycopeptide (16). Oligosaccharide 15 eluting at 15- 17 minis a disialyl decasaccharide of structure previously reported (14). Other minor compounds have not been specificially identified as yet. Of interest, with this solvent elution system is the finding that free N-acetylneuraminic acid elutes at 6.6 min compared to the higher molecular weight hexasaccharide which elutes at 5 min. However, with sialyl oligosaccharides in which the sialic acid is glycosylated these compounds are eluted in order of molecular weight. Thus, neuraminyllactose, a trisaccharide eluted at 3.8 min. However, the hplc system we used would not resolve the microheterogeneity of linkages present in sialyl oligosaccharides. Figure 4 gives the hplc profile of urine from a Type 2 sialidosis (15) patient with dysmorphic features and severe mental retardation. In Type 1 patients (normosomatic group, see Ref. (15)) the hplc profile -is similar except that in this

High-pressure liquid chromatography using a PBondapaWcarbohydrate column (a Waters Associates proprietary-bonded silica with an amine functionality) has great potential in analyses of glycoconjugates derived from glycoproteins. As shown here, hplc gives diagnostic elution profiles of liver and urinary oligosaccharides and glycopeptides in a number of lysosomal storage diseases. It can also be used for the preparation of small amounts of pure compounds. Thus, up to 100 pg of each major oligosaccharide can be isolated in a single run (maximum time 15-20 min) from a small aliquot of a 24-h urine sample from GM,-gangliosidosis Type 1 patients. With the availability of semimicro-methods of analyses such as Fourier transform nuclear magnetic resonance spectroscopy (5) and combined gas-liquid chromatographic-mass spectroscopic determination in permethylation analyses of oligosaccharides (I), the hplc method offers a rapid method for the isolation of homogeneous oligosaccharides for complete structure determinations. The method also allows for rapid checking of the purity of fractions obtained during large-scale Bio-Gel or Sephadex chromatography of oligosaccharides and glycopeptides. One disadvantage of the method described here is the low sensitivity of the differential refractometer detector. It was found that lo-30 pg of oligosaccharides was necessary to obtain satisfactory responses. Further, the refractometer detector cannot be used with gradient elutions. However, borohydride-reduced oligosaccharides give similar

OLIGOSACCHARIDES

AND GLYCOPEPTIDES

elution characteristics to the free-reducing oligosaccharide (Figs. 1 and 2). The detection of very small amounts of oligosaccharides could therefore be achieved if labeled with tritium by sodium borotritiide reduction. The rapid separation of radiolabeled compounds by hplc would be useful in biochemical studies involving carbohydrates of mammalian systems such as analysis of products of glycosylhydrolase and glycosyltransferase reactions.

IN STORAGE

219

DISORDERS

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ACKNOWLEDGMENTS We thank Dr. Robert Desnick, Department of Pediatrics and Genetics, Mount Sinai School of Medicine, New York, for the urine from mannosidosis patients; Dr. H. Schacter, Hospital for Sick Children, Toronto, for the urine from an aspartylglucosaminuria patient; Dr. M. Vanasse and Dr. A. Larbrisseau, HBpital Ste-Justine, Montreal, for the urine from G,,-gangliosidosis and fucosidosis patients: and Dr. J. A. Lowden for the urine from G,,-gangliosidosis and sialidosis Type 2 patients. Mrs. P. Skelton provided expert technical assistance. This research was supported by a Medical Research Council of Canada Grant MT-1345 to L.S.W.

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