Sulfated N-linked carbohydrate chains in porcine thyroglobulin

Volume 241, number 1,2, 246-250

FEB 06523

December 1988

Sulfated N-linked carbohydrate chains in porcine thyroglobulin Johannis P. Kamerling, I r m a Rijkse, Augustinus A.M. Maas, J. Albert van K ui k and J ohannes F.G. Vliegenthart Department o f Bio-Organic Chemistry, Utrecht University, Transitorium 111, PO Box 80.075, 3508 TB Utrecht, The Netherlands

Received 16 September 1988 N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc~2~6Galfll~4GlcNAcfll--.2MancO--,3[(SOaNa--.3)Galfll--,4GlcNAcfll--.2. Man~ 1~6]Manfll ~4GlcNAcfll ~4[Fuc~ I --*6]GlcNAc-ol and NeuAc~2 ~6Galfll --.4(SO3Na~6)o_ 1GlcNAcfll --*2Man~ I ~ 3[NeuAc~2 ~ 6Galfll --*4(SO3Na --*6)l_oGlcNAcfll - . 2Man~ 1---,6]Manfll --*4GlcNAcfl 1--*4[Fuc~ 1---,6]GlcNAc-ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity. Thyroglobulin; Sulfated N-linked carbohydrate; (Porcine)

1. INTRODUCTION Thyroglobulin, the major iodinated glycoprotein synthesized in the thyroid gland, is the polypeptide precursor of the thyroid hormones. Analysis of the carbohydrate chains of thyroglobulin (molecular mass -660 kDa) from several species has demonstrated that mainly two types of chains occur, generally called unit-A type (oligomannose type) and unit-B type (Nacetyllactosamine type) [1-10]. For human thyroglobulin also unit-C type (mucin type) [4] and unit-D type (proteoglycan type) [11] chains have been indicated. In many cases c~l ,3Gal residues can form part of the unit-B type chains [12-14]. Concerning the N-linked oligosaccharide chains of porcine thyroglobulin [5,6], the literature data indicate the presence of Mans_9GlcNAc2 structures, whereby the trimming of Man residues

seems to take place randomly [7]. For the Nacetyllactosamine type a series of partially sialylated di- (75%) and tri- (25°7o) antennary structures with an crl ,6-1inked Fuc residue at the Asn-bound GlcNAc unit have been reported [8]. In addition, a terminal Gakel ,3Gal~l ,4 element in the Mano~l---% branch of a monosialylated (oz2 ,6-1inked NeuAc; Manc~1--~3 branch) diantennary structure has been shown to occur [14]. The recent finding of phosphate [10,15-17] and, especially, sulfate [18] groups in thyroglobulins from different biological origins prompted us to report data on the structural identification of sulfated N-linked unit-B type carbohydrate chains in porcine thyroglobulin.

2. MATERIALS AND METHODS 2.1. Porcine thyroglobulin

Correspondence address: J.P. Kamerling, Department of BioOrganic Chemistry, Utrecht University, Transitorium III, PO Box 80.075, 3508 TB Utrecht, The Netherlands

Porcine thyroglobulin was obtained from Sigma. SDSpolyacrylamide gel electrophoresis gave the usual pattern of two main bands, together with three faint bands of lower molecular mass [19,20]. Its sugar composition (Fuc/Man/Gal/GlcNAc/

Published by Elsevier Science Publishers B. V. (Biomedical Division)

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00145793/88/$3.50 © 1988 Federation of European Biochemical Societies

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NeuAc = 0.4 : 3.0: 1.2 : 2.6: 0.4) and carbohydrate content (7%) are in accordance with literature data [1,2,21]. 2.2. Preparation of oligosaccharide-alditols The hydrazinolysis procedure on porcine thyroglobulin (six 125-mg portions) followed by high-voltage paper electrophoresis was carried out essentially as described [22,23]. The oligosaccharide-alditols were recovered from the paper by elution with water, yielding one neutral and three acidic fractions. The acidic fractions were further subfractionated by HPLC on Lichrosorb-NH2 using elution systems of acetonitrile/ 15-30 mM KHzPOn-K2HPO4, pH 5.2 [24], and monitored at 205 nm. Fractions were desalted on Bio-Gel P-2 (200-400 mesh) using water as eluent and refractive index detection. 2.3. Monosaccharide analysis Carbohydrate samples were subjected to the methanolysis procedure and analyzed by GLC on CP Sil 5, as described [25]. 2.4. Methylation analysis Methylation analysis was carried out essentially as described [26]. Permethylated material was hydrolyzed with 4M trifluoroacetic acid, the obtained mixtures of partially methylated monosaccharides were reduced with NaB2H4 in water, and the partially methylated alditol acetates analyzed by GLC-MS. 2.5. 500-MHz JH-NMR spectroscopy Oligosaccharide-alditols were repeatedly exchanged in 2H20 (99.96 atom% EH) w i t h intermediate lyophilization. Resolution-enhanced 500 MHz ~H-NMR spectra were recorded in 2H20 at 27°C on a Bruker WM-500 spectrometer (SON hfNMR facility, Department of Biophysical Chemistry, University of Nijmegen, The Netherlands). Chemical shifts (8) are given relative to sodium 4,4-dimethyl-4-silapentane-l-sulfonate, but were actually measured indirectly to acetone (,5 = 2.225 ppm) [27]. 3. R E S U L T S I n the f r a m e w o r k o f o u r N M R studies o n sulfated m o n o - [28] a n d oligo- [29,30] saccharides, p o r c i n e t h y r o g l o b u l i n was investigated for the presence o f sulfated N - l i n k e d c a r b o h y d r a t e chains. T h e hydrazinolysis p r o c e d u r e in c o m b i n a t i o n with high-voltage paper electrophoresis yielded one n e u t r a l (N) a n d three acidic (A1, A2, A3) fractions in the m o l a r ratio o f 27 : 35 : 28 : 10, respectively. As A 2 a n d A3 t u r n e d out to c o n t a i n the searched m a t e r i a l , a t t e n t i o n will o n l y be paid to these fractions in this paper. S u b f r a c t i o n a t i o n o f A2, isolated f r o m the ' d o u b l e - n e g a t i v e l y - c h a r g e d ' region of the paper e l e c t r o p h e r o g r a m , o n L i c h r o s o r b - N H z yielded a series of peaks, o f which A 2 b is o f interest. The 1 H - N M R s p e c t r u m of A 2 b shows the presence of

December 1988

o n e m a j o r c o m p o u n d . The N M R features o f this oligosaccharide-alditol, presented in table 1, resemble those of reference c o m p o u n d IgM, which is a classical d i a n t e n n a r y structure t e r m i n a t e d with ~1 ,4-1inked Gal in the M a n a l ,6 b r a n c h a n d or2 ,6-1inked N e u A c in the Mantel ,3 b r a n c h a n d having a n eel ,6-1inked L-fucose residue at the A s n - b o u n d N - a c e t y l g l u c o s a m i n e [31]. H o w ever, of the s t r u c t u r a l - r e p o r t e r - g r o u p signals the G a l - 6 ' H-1 d o u b l e t had shifted d o w n f i e l d f r o m = 4.470 p p m to c~ = 4.587 p p m . F u r t h e r m o r e , in c o n t r a s t to the s p e c t r u m o f IgM, in that of A2b two a d d i t i o n a l signals can be observed in the structural-reporter-group region, namely, a d o u b l e t of doublets at ~; = 4.339 p p m (J values of 3.2 Hz a n d 10.0 Hz) a n d a d o u b l e t of doublets at 8 = 4.294 p p m (J values o f 3.2 Hz a n d - 1 Hz). T h e latter r e s o n a n c e has the typical shape o f a Gal H-4 signal. By selective 1H-decoupling o f the signal at ~; = 4.339 p p m , c o n n e c t e d signals at ~ = 4.294 p p m a n d ~; = 3.684 p p m are f o u n d by difference spectroscopy. I n the same way, selective ~H-decoupling o f the H-1 signal o f G a l - 6 ' (6 = 4.587 p p m ) a n d difference spectroscopy identified the r e s o n a n c e at 6 = 3.684 p p m as G a l - 6 ' H-2. W h e n a similar e x p e r i m e n t is carried out for the H-1 signal of Gal-6, the c o r r e s p o n d i n g H-2 r e s o n a n c e is f o u n d at d; = 3.534 p p m . I n conclusion, the signals at ~ = 4.339 p p m a n d d; = 4.294 p p m can be a t t r i b u t e d to G a l - 6 ' H-3 a n d H-4, respectively. Since the m a j o r c o m p o u n d in s u b f r a c t i o n A 2 b is a m o n o s i a l y l a t e d structure, a n a d d i t i o n a l acidic s u b s t i t u t i o n has to be present. I n view o f the literature d a t a o n acidic c a r b o h y d r a t e chains, it is o b v i o u s to suppose that either a p h o s p h a t e or a sulfate g r o u p is involved [10,15-18]. The most obvious positions o f a t t a c h m e n t are G a l - 6 ' C-3 or C-4, as can be c o n c l u d e d f r o m the d o w n f i e l d positions of G a l - 6 ' H-3 a n d H-4. Because o f the absence o f 31p couplings o n these signals, the occ u r r e n c e o f p h o s p h a t e can be excluded. I n the case o f sulfate, the chemical shift values at d; = 4.339 p p m a n d d; = 4.294 p p m were c o m p a r e d to those o f sulfated m o n o s a c c h a r i d e references. I n table 2, the 1H-NMR d a t a o f the methyl glycosides o f ce-D-galactopyranose, B - D - g a l a c t o p y r a n o s e , 3-O-SO3Na-ce-D-galactopyranose a n d 4-O-SO3Nace-D-galactopyranose [28] are s u m m a r i z e d . G o i n g f r o m methyl a - D - g a l a c t o p y r a n o s i d e to methyl 247

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Table 1 IH chemical shifts of structural-reporter-group protons of the constituent monosaccharides of fractions A2b and A3c obtained from porcine thyroglobulin, together with those of reference compounds lgM and PT Reporter group

Residuea

Chemical shiftsb in c A2b

IgM

A3ca

PT

S6

H1

GlcNAc-2 Man-3 Man-4 Man-4' GlcNAc-5 GIcNAc-5 ' Gal-6 Gal-6' Fuc

4.713 4.778 5.136 4.924 4.607 4.580 e 4.444 4.587 e 4.898

4.714 4.760 5.136 4.924 4.605 4.581 4.445 4.470 4.896

n.d. n.d. 5.139 4.942 4.602 4.643 4.445 4.486 4.896

4.715 4.786 5.135 4.942 4.605 4.605 4.443 4.443 4.896

H-2

GlcNAc-l-ol Man-3 Man-4 Man-4'

4.213 4.255 4.198 4.108

4.220 4.258 4.197 4.110

4.212 4.258 4.205 4.112

4.219 4.260 4.198 4.114

H-3a H-3e H-3

NeuAc NeuAc Gal-6'

1.718 2.669 4.339

1.720 2.668 n.d.

1.719 f 2.671 f n.d.

1.718 f 2.669/2.673 n.d.

H-4

Gal-6'

4.294

n.d.

n.d.

n.d.

H-5

Fuc

4.072

4.071

4.073

4.072

H-6 H-6'

GIcNAc-5 ' GIcNAc-5 '

n,d. n.d.

n.d. n.d.

4.434 4.314

n.d. n.d.

CH3

Fuc

1.223

1.224

1.225

1.224

NAc

GlcNAc-l-ol GlcNAc-2 GlcNAc-5 GlcNAc-5' NeuAc

2.056 2.089 2.070 2.048 2.030

2.057 2.088 2.071 2.048 2.031

2.056 2.089 2.072 2.066 2.030 f

2.056 2.090 2.071 2.065 2.030 f

a For numbering of the monosaccharide residues, see text b Chemical shifts are given for neutral solutions at 27°C, in ppm downfield from internal 4,4-dimethyl-4-silapentane-l-sulfonatein 2H20, acquired at 500 MHz (but were actually measured relative to internal acetone: ~ = 2.225 ppm) c Structures are represented by short-hand notation [27]: o, GlcNAc; * , Man; N, Gal; o, NeuAca2----*6; r~, Fuc; S, sulfate d For reasons of simplicity, the d values for GIcNAc-5/5' H-1 and Gal-6/6' H-1 have been grouped for a sulfate group at GIcNAc-5' e Assignments may have to be interchanged f Signal stems from two NeuAc residues

3-O-SO3Na-ce-D-galactopyranoside, similar downf i e l d s h i f t s f o r H - 3 a n d H - 4 (Ad; = + 0 . 6 6 6 p p m a n d z18 = + 0 . 3 5 6 p p m , r e s p e c t i v e l y ) w e r e o b s e r v e d , as g o i n g f r o m m e t h y l f l - D - g a l a c t o p y r a n o s i d e t o G a l - 6 ' in s u b f r a c t i o n A 2 b ( H - 3 , zl~ -+ 0 . 6 9 5 p p m ; H - 4 , A~ = + 0 . 3 7 2 p p m ) . I n t h e c a s e of the 4-O-sulfated analog quite a different NMR 248

p e a k p a t t e r n w a s o b s e r v e d . It h a s t o b e n o t e d t h a t similar positions for Gal H-3 and H-4 have recently b e e n r e p o r t e d f o r a t e r m i n a l 3 - s u l f a t e d G a l fll , 4 r e s i d u e in a n O - l i n k e d c h a i n [32]. S u m marizing, the terminal Gal r e s i d u e in t h e Mantel , 6 a n t e n n a ( G a l - 6 ' ) in A 2 b is s u l f a t e d at position C-3, leading to the following structure:

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FEBS LETTERS 6'

5'

4'

SO3Na----* 3 G a l f l l ----~ 4GlcNAc/5'I ~

FucoH - - - * 6

2Manor I ~ 6

... /

NeuAca2 ~

6Gal~'l ~

4GlcNAc~'I ~

6

2Mance 1 ~

5

1H-NMR data for the methyl glycosides of ce-Dgalactopyranose, B-D-galactopyranose, 3-O-SO3Na-o~-Dg a l a c t o p y r a n o s e and 4 - O - S O 3 N a - c r - D - g a l a c t o p y r a n o s e [28]

H-I H-2 H-3 H-4 H-5 H-6 H-6' OMe

Manfll ~

3

3

4 G l c N A c f l l ---* 4 G l c N A c - o l

2

1

4

Table 2

Protons

December 1988

Chemical shifts a in o~-D-Gal

3-O-SO~Naa-D-Gal

4-O-SO3Nacr-D-Gal

fl-D-Gal

4.837 3.819 3.811 3.968 3.897 3.741 3.752 3.414

4.899 3.982 4.477 4.324 3.935 3.751 3.759 3.430

4.867 3.855 3.941 4.713 4.030 3.810 3.755 3.425

4.315 3,501 3,644 3,922 3.695 3.751 3.796 3.573

Chemical shifts are given at 27°C, in ppm downfield from internal 4,4-dimethyl-4-silapentane-l-sulfonate in Z H 2 0 , acquired at 500 M H z (but were actually measured relative to internal acetone: d = 2 . 2 2 5 ppm)

Subfractionation of fraction A3, isolated from the 'triple-negatively-charged' region of the paper electropherogram, on Lichrosorb-NHz resulted in several peaks, of which only fraction A3c is of interest. The tH-NMR spectrum of subfraction A3c shows a nearly pure compound. Comparison of the various resonances in the anomeric region of

the spectrum as well as in the region ~ < 2.8 ppm, with the spectral features of a classical c~2 ,6 disialylated diantennary carbohydrate chain with an cel--+6-1inked Fuc residue at the Asn-bound GIcNAc unit, denoted PT, shows that the basic structure of A3c is similar to PT. The intensities of the GIcNAc-5/5' and Gal-6/6' H-1 doublets in A3c have only half the intensities of those of the corresponding signals in PT. But additionally, for GIcNAc as well as for Gal, a more downfield doublet is observed, namely, at ~ -- 4.643 ppm and at d = 4.486 ppm, respectively. This means that in one of the antennae a structural element has changed, as compared to PT. Furthermore, two additional non-anomeric downfield signals, resonating at ~ = 4.314 ppm (Jvalues of 4.8 Hz and 11.0 Hz) and at d = 4.434 ppm (J values of 1.2 Hz and 11.0 Hz) are observed. By selective irradiation it was found that both resonance patterns are coupled with each other. In view of earlier reports [29,33], such a typical coupled pattern belongs to H-6 and H-6' of one of the monosaccharide residues, bearing a sulfate group at C-6. Methylation analysis of fraction A3c yielded a 4,6-disubstituted GIcNAc residue, indicating that GlcNAc-5 or GIcNAc-5' bears a sulfate group at C-6. Combining the various data, the following two structures for A3c are possible:

( S O ~ N a - - - + 6 ) l_0

\,

\\ x

6'

5'

NeuAco~2---+ 6 G a l f l l ~ 4 G l c N A c f l l

4' ----, 2ManoL 1 ----*6

Fuco~ 1 ----*6 -x.

\ M an~5'l ~ 3

NeuAcce2---, 6Ga~l ~

4 G l c N A c f l l ---* 2Mance 1 ~

6

/

/

/

/

/

/

5

4GIcNAc~'I ~ 2

4GIcNAc-ol l

3

4

(SO3Na---%)o_/

249

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Also the occurrence of a mixture of both structures c a n n o t be e x c l u d e d so far. T h e r e c e n t l y r e p o r t e d c~ values of GlcNAc H-6 and H-6' in t h e Galfll ,4(SO3Na----*6)GlcNAc element, present in a n O - l i n k e d c h a i n , a r e in t h e s a m e r a n g e as t h e v a l u e s i n c l u d e d h e r e [34]. R e c e n t studies o n carb o h y d r a t e c h a i n s l i b e r a t e d f r o m v a r i o u s 35Sl a b e l e d m a m m a l i a n cell lines s u g g e s t e d t h e o c c u r rence of a NeuAccr2 ,6/3Galfll----*4(SO3Na , 6 ) G I c N A c e l e m e n t in N - l i n k e d o l i g o s a c c h a r i t i e s [35].

Acknowledgements: This investigation was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO), and by the Netherlands Foundation for Cancer Research (KWF, grant UUKC 83-13).

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