The murine sublingual and submandibular mucins their isolation and characterization

432

Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 4 3 2 - - 4 4 0 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27856

THE MURINE SUBLINGUAL AND S U B M A N D I B U L A R MUCINS T H E I R ISOLATION AND C H A R A C T E R I Z A T I O N

P.A. R O U K E M A *, C.H. O D E R K E R K a n d M.S. S A L K I N O J A - S A L O N E N **

Department of Oral Biochemistry, Vrije Universiteit, Amsterdam. P.O. Box 7161 (The Netherlands) (Received September 18th, 1975)

Summary From the mouse sublingual and submandibular glands high.molecular weight glycoproteins (mucins) were isolated. These mucins appeared to be homogeneous in polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. S20,w values of 10.9 and 5.5 were found for the sublingual and submandibular mucin respectively. With sodium dodecyl sulfate or N-acetylcysteine no subunits could be detected. Both mucins consisted for a b o u t 1/3 of protein and 2/3 of carbohydrate. Their mucin character was also denoted by the high conter~t of serine plus threonine. Respectively 42 mol% and 34 mol% of the protein core of the sublingual and submandibular mucins consisted of these amino acids. The main sugars in these mucins were sialic acid, galactosamine, galactose, glucosamine and mannose. The molar ratio for the sublingual and submandibular mucin being 1.00 : 1.03 : 1.08 : 0.26 : 0.23 and 1.00 : 0.71 : 1.10 : 0.65 : 0.53, respectively. The sialic acid content of both mucins was a b o u t 25%. Fucose and sulfate, on the other hand, were less than 1%. The presence of sulfate was also indicated b y preliminary studies in vivo on the incorporation of [3sSO4] sulfate.

Introduction The histochemical characterization of the mucins from the salivary glands of different species has been described b y several investigators [ 1--3 ]. On the base of staining reactions with periodic acid-Schiff reagent, colloidal iron, Alcian * Address for c o r r e s p o n d e n c e .

** Present address: I n s t i t u t e o f M i c r o b i o l o g y , Uni~;ersity of Helsinki, Malminkato 20, Helsinki 10, Finland.

433

blue, treatment with sialidase and mild acid hydrolysis, indications were obtained for the occurrence of sialo-, fuco- and sulfomucins. In the mouse, as in other rodents, the predominantly mucous sublingual glands seem to contain mainly sialomucins, while no indications were obtained for the presence of sulfomucins. The seromucous submandibular glands are supposed to contain sialo- as well as fucomucins. The precise localization of these c o m p o u n d s in the glands is not known. Dische et al. reported that the sialic acid to fucose ratio in the submandibular saliva from dog [4,5] and cat [6] is different after sympathetic and parasympathetic stimulation. This m a y indicate that the sialo- and fucomucins are partly present in different cell types, the variation in the sialic acid to fucose ratio being determined b y the nature of the stimulus, eliciting the secretion. On the other hand L o m b a r t and Winzler [7] found that more than one type of oligosaccharide chain may be present in the same mucin. These authors suggest that the nature of the stimulus, which is known to change the fucose to sialic acid ratio in the canine submandibular secretions, would affect the relative activities of different glycosyl transferases, to a c o m m o n substrate. In order to get more insight into the question h o w a stimulus affects the secretion process, it is necessary to know the cellular localization and chemical composition of the mucins, involved in the secretory processes. In this paper the isolation and characterization of the mucins from the murine sublingual and submandibular glands will be described.

Experimental

Isolation of mucins. Female mice {strain Swiss-random) were allowed to feed and to drink ad libitum overnight. At six o'clock in the morning the food was removed. The animals were killed 8 h later b y decapitation. By this procedure optimal filling of the glands was obtained. The glands were removed rapidly at room temperature and dissected o u t under the microscope at 4°C. If mice of 15--20 g are used the sublingual and submandibular glands of the mouse can be easily separated and cleaned. This is in contrast to the finding for the rat glands [ 2 1 ] . In general 30 glands were used for the isolation of the mucins. With only a few modifications, the isolation procedure described b y Oemrawsingh and R o u k e m a [9] for the preparation of human submandibular mucins, has been used. The sublingual and submandibular glands were homogenized in a PotterElvehjem homogenizer with a Teflon pestle in 19 parts of distilled water. After centrifugation at 38 000 X g for 20 min the supernatant (S,) was adjusted to pH 5.0 with 0.1 M sodium acetate buffer and subsequently heated at 100°C for 30 min. This procedure resulted in the precipitation of part of the protein material. The precipitate was removed by centrifugation, first at 38 000 × g for 15 min and then at 100 000 X g for 1 h (MSE-50-centrifuge). The somewhat opalescent supernatant ($2) was dialyzed against distilled water with several changes of the water during 6 h and concentrated in a rotating evaporator to a b o u t 1.5 ml.

434

The concentrated material was separated on a Biogel P-300 column (length 30 cm, diameter 1.5 cm), equilibrated with 0.154 M sodium chloride. Elution was carried out with the same medium at 4°C. Fractions of 1.5 ml were collected at a flow rate of 1--3 ml/h. The void volume fractions which contained the mucin, were combined, dialyzed against distilled water overnight and freezedried. Sedimentation velocity of the mucins. The S values were determined in 0.1052 M sodium chloride + 0.0144 M Na2HPO4 + 0.0563 M NaH2PO4, pH 7.15 (ref. 11). The experiments were performed in a Beckman Spinco analytical ultracentrifuge with the Schlieren optic at 42 040 rev./min in a 12 mm double sector cell.

The effect o f N-acetylcysteine and sodium dodecyl sulfate on the behaviour o f the mucins. Their effect on the chromatographic behaviour of the mucins was studied on Biogel P-300. In the analytical ultracentrifuge the presence of subunits was investigated. For the chromatographic investigation the mucins were preincubated with (a) a solution of 0.05 M N-acetylcysteine {adjusted to pH 8.0) in 0.154 M sodium chloride at 37°C for 60 min, or (b)1% dodecyl sulfate in 0.154 M sodium chloride + 0.01 M sodium phosphate buffer, pH 7.0 at 20°C for 2 h, or (c) the combination of a) and b), at 37°C for 1 h. The equilibration of the Biogel P-300 column and the elution of the column was done with the same buffers. The only difference being that N-acetylcysteine was 0.005 M instead of 0.05 M. To study the sedimentation pattern, the samples were preincubated with 0.2% sodium dodecyl sulfate and/or 0.05 M N-acetylcysteine in the buffer, described for the analytical ultracentrifuge. In all cases 8 • 10 -s M sodium azide was added to prevent bacterial growth. Analytical procedures. The amino acids were analyzed with a Beckman Unichrom analyzer after hydrolysis in 6 M HC1 at l l 0 ° C for 24 h. The protein c o n t e n t of the samples was measured according to Lowry et al. [12] with bovine serum albumin as a reference and was also calculated from the sum of the amino acid values. Total hexosamine was determined as described by Ruinen et al. [13] after hydrolysis in 2 M HC1 at 100°C for 4 h. Glucosamine and galactosamine as well as their ratio were determined from a shortterm run on the amino acid analyzer after hydrolysis under the same conditions. Hexose was assayed by the orcinol m e t h o d [14]. The different hexoses were determined quantitatively by GLC [15] and identified by TLC. The samples were hydrolysed in 2 M HC1 at 100°C for 4 h. Ascending chromatography on silica gel plates was carried out with the solvent system ethylacetate/propan-1ol/propan-2-ol/water (8 : 5 : 1 : 1, v/v). After 16 h the plates were dried, coloured with aniline phosphate and heated at l l 0 ° C for 10 min. Sialic acid was determined according to Warren [16] and fucose by the m e t h o d of Dische and Shettles [17]. Sulfate was assayed by the chloranilate procedure, as described by Spencer [ 18]. Polyacrylamide gel electrophoresis. Electrophoresis was carried out in Tris • HC1 buffer, pH 8.9 as described by Rainer Maurer [19] and Oemrawsingh and R o u k e m a [9] ; 3.75% of acrylamide with 0.2% of bisacrylamide were used. All solutions contained 0.1% sodium dodecyl sulfate (w/v).

435

Results

The purification The mean weight of the dissected sublingual and submandibular glands was 8.2 and 36.5 mg, respectively. The sialic acid content was very high in the sublingual glands (12.1 (10.5--14.0) mg/g wet weight) in comparison to the submandibular glands (2.54 (2.32--2.99) mg/g wet weight). Respectively 90 and 63% of this sialic acid was present in the soluble fraction $1 (see Table I). A major step in the purification of the mucins was the heating at 100°C during 30 min {Table I). This was done at pH 5.0 instead of pH 6.0 as has previously been described for the human submandibular mucins [ 9 , 1 0 ] , because optimal results for the murine mucins were obtained at pH 5.0. Precipitation of the denaturated components by centrifugation resulted in a four-fold purification for the sublingual mucin and about a two-fold purification for the submandibular mucin. The chromatography on Biogel P-300 is shown in Fig. la and l b for the sublingual and the submandibular gland extracts respectively. The Biogel P-300 passage was the determining purification step for the submandibular gland mucin. The protein/sialic acid ratio diminished by a factor of about 25. In this case the bulk of the protein and half of the sialic acid were recovered from the second peak (see Fig. lb). The recovery of sialic acid from the Biogel P-300 column was 84 and 74% respectively for the sublingual and submandibular gland extracts. This recovery could be enhanced to about 100% by the addition of N-acetylcysteine to the sample and the elution solvent. This addition strongly

TABLE I PURIFICATION

OF THE MOUSE SUBLINGUAL

AND SUBMANDIBULAR

Sublingual gland m u c i n * Protein/ sialic acid ratio ( w / w ) Homogenate c e n t r i f u g e at 38000X g for 30 min S u p e r n a t a n t (S 1 ) h e a t at l O 0 ° C at p H 5 . 0 f o r 3 0 rain, c e n t r i f u g e at 38000X gfor 1 5 rain, t h e n at 1 0 0 0 0 0 × g for 60 min S u p e r n a t a n t (S 2 ) Biogel P - 3 0 0

Passage M u c i n (void v o l u m e peak)

Recovery sialic acid (%)

7.09

GLAND MUCINS

S u b m a n d i b u l a r gland m u c i n * Purification factor * *

Protein/ sialic acid ratio ( w / w )

Recovery sialic acid (%)

Purification factor * *

1.0

47.8

--

1.0

4.34

100

1.63

37.7

100

1.27

1.12

82

6.33

22.5

81

2.12

0.72

69

9.84

* Mean of 5 e x p e r i m e n t s . ** E x p r e s s e d as t h e i n c r e a s e in t h e sialic acid t o p r o t e i n ratio.

0.86

60

55.6

436 800--

MSL

--

MSM ~t

T

~soo-

I

.~

400--

~

-

o200--

iI

l

Vo

.......... ,6, 2o, a

I

Vo

I

4

i

I 12

s

16

I I 20 24 fraction no.

b

Fig. 1. P u r i f i c a t i o n of t h e s u b l i n g u a l m u c i n (a) a n d t h e s u b m a n d i b u l a r m u c i n (b) o n Biogel P - 3 0 0 ( c o l u m n l e n g t h 3 0 c m , d i a m e t e r 1.5 c m ) , e q u i l i b r a t e d a n d e l u t e d w i t h 0 . 1 5 4 M s o d i u m c h l o r i d e . F r a c t i o n s o f 1.5 m l w e r e c o l l e c t e d at a flow r a t e of 1--3 m l / h . The void v o l u m e f r a c t i o n s c o n t a i n e d t h e m u c i n s .

reduced the high viscosity of the sublingual gland sample. However, N-acetylcysteine, sodium dodecyl sulfate and the combination of both (see methods) did neither change the elution pattern of the applied extracts nor that of the purified rechromatographed mucins. In the analytical ultracentrifuge the mucins were studied at two concentrations (Table II). A dependence of the S value on the concentration of the mucin was only clearly indicated for the sublingual mucin. The sedimentation pattern and the S values were either not at all or hardly influenced after preincubation with 0.2% sodium dodecyl sulfate (w/v) or 0.05 M N-acetylcysteine. In all cases only a single peak could be detected. So, under the conditions of assay, used by us, no indications for the existence of subunits were found. The higher S20,w values for sublingual mucin in comparison to the submandibular mucin points to a higher molecular weight for the sublingual mucin. This is consistent with a much lower migration rate of the sublingual mucin in polyacrylamide gel electrophoresis. (Fig. 2).

The carbohydrate and amino acid analysis The chemical composition of the sublingual and submandibular mucins have been presented in the Tables III and IV. The sugar c o m p o n e n t s in the sublingual and the submandibular mucin were sialic acid, galactosamine, glucosamine, galactose, mannose and fucose. The ratios of galactosamine/glucosamine and galactose/mannose were different for both mucins (Table III). The level of fucose was very low in both cases. The sulfate content was also very small. This means that both mucins must be classified as sialomucins.

437 T A B L E II S E D I M E N T A T I O N V E L O C I T Y V A L U E S OF T H E M U R I N E S U B L I N G U A L AND S U B M A N D I B U L A R MUCINS

Mucin

Mucin conc.

Incubation conditions *

Sublingual m u c i n

1.5 m g / m l 3.5 m g / m l

Buffer Buffer B u f f e r + 0.2% s o d i u m d o d e c y l sulfate B u f f e r + 0.2% s o d i u m d o d e c y l sulfate + 0 . 0 5 M N-acetylcysteine

10.9 6.7

Buffer Buffer B u f f e r + 0.2% s o d i u m d o d e c y l sulfate B u f f e r + 0.2% s o d i u m d o d e c y l sulfate + 0.05 M N-acetylcysteine

4.9 5.5

Submandibulax mucin

5.0 m g / m l 2.5 m g / m l

$ 2 0 , w values

6.8

6.8

5.4

5.5

* Buffer: 0 . 1 0 5 2 M N a C l / 0 . 0 1 4 4 M N a 2 H P O 4 / 0 . 0 5 6 3 M N a H 2 P O 4 , p H 7 . 1 5 .

Z

Fig. 2. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f t h e s u b l i n g u a l m u c i n a n d s u b m a n d i b u l a r m u c i n in 3 . 7 5 % a c r y l a m i d e at p H 8.9. All s o l u t i o n s c o n t a i n e d 0 . 1 % s o d i u m d o d e c y l sulfate ( w / v ) . 8 0 ~g o f m u c i n w e r e a p p l i e d p e r gel. T h e r u n n i n g t i m e was 75 rain at a c o n s t a n t c u r r e n t of 3 m A / g e l . S u b l i n g u a l m u c i n : gels 1 a n d 2. S u b m a n d i b u l a r m u c i n : gels 3 a n d 4. Gels 2 a n d 4 w e r e s t a i n e d w i t h A m i d o Black, gels 1 a n d 3 w i t h the periodic aeid-Schiff (PAS) reagent.

438 TABLE

III

CHEMICAL

COMPOSITION

Component

Sialic acid a Hexosamine a GalNAc

Protein

b

MUCINS

Mol~Lr r a t i o

Sublingual

Submandibulax

Sublingual

Submandibular

mucin

mucin

mucin

muein

25.9 18.8 15.4

25.6 19.3 10.1

1.00 -1.03

1.00 -0.71

3.4

9.2

0.23

0.65

20.1 16.2

23.6 15.9

-1.08

-1.10 0.53

Man Fucose

AND SUBMANDIBULAR

Relative weight (%)

GlcNAc Hexose Gal

OF THE SUBLINGUAL

3.9

7.7

0.26

0.S

0.6

0.05

0.07

34.1

30.9

--

--

0.4

0.5

--

--

Sulfate

a S i a l i c a c i d is e x p r e s s e d a s N - a c e t y l n e u r a m i n i c

acid and the hexosamines

as t h e N - a c e t y l d e r i v a t i v e s .

b By a m i n o a c i d a n a l y s i s .

Their amino acid composition was very typical (Table IV). In the sublingual mucin the level of Glu + Asp and Arg + His + Lys is low, whereas Set and Thr are very high (20.4 and 21.2 mol%, respectively). Ala had also a high level, viz. 17.6 mol%. In the submandibular mucin on the other hand Ser is much lower than Thr (10.8 and 23.2 mol%, respectively).

TABLE

IV

AMINO

ACID COMPOSITION

OF THE SUBLINGUAL

A m i n o acid

Sublingual m u c i n * (mol/lO0 tool)

Ala

17.6 ± 1.56

Asp Cys Gin Gly Ile Leu Met Phe Pro Set Thr Tyr Val Arg His Lys

3.6 0.6 5.1 9.7 1.7 2.8 1.6 1.4 5.7 20.4 21.2 0.8 3.7 2.0 0.5 1.6

± 0.62 ± 0.41 ± 0.71 ± 0.68 ± 0.40 + 0.68 ± 0.71 + 0.36 + 1.62 + 1.12 -+ 1 . 5 6 -+ 0 . 2 5 + 0.38 -+ 0 . 2 4 + 0.21 ± 0.40

* Mean of 5 experiments

± S.E.

AND SUBMANDIBULAR

Submandibular m u c i n * (mol/lO0 mol) 9.2 + 0.52 8.9 0.3 6.6 5.0 4.0 4.7 0.7 2.3 9.3 10.8 23.2 1.6 3.4 2.9 0.9 6.3

± 0.54 ± 0.20 ± 0.94 ± 0.70 ± 0.29 ± 0.74 ± 0.08 ± 0.24 ± 1.29 ± 0.76 -+ 2 . 7 1 +- 0 . 1 7 ± 0.61 + 0.29 ± 0.24 ± 0.24

MUCINS

439 Discussion In most cases the m e t h o d of Tettamanti and Pigrnan [20] which is based on clot formation by cetavlon and subsequent fractional precipitation with ethanol, or a comparable m e t h o d has been employed for the purification of glandular mucins. However, difficulties are encountered, when very small amounts of mucin must be isolated from a large a m o u n t of contaminating protein [21 ]. In that case the m e t h o d used by us (refs. 9, 10 and this paper) may be more convenient. From the sialic acid c o n t e n t of the soluble cell fraction (S~, see Table I), and the composition of the mucins, it can be calculated that about 4% of the wet weight of the mouse sublingual gland consists of mucin. For the mouse submandibular gland this figure is less than 0.4%. Nevertheless the submandibular mucin could also be readily purified. Both mucins contain about 25% of sialic acid. A comparable figure was obtained for the main rat sublingual mucin by Moschera and Pigman [21]. Keryer et al. [22] found somewhat lower values for a rat submandibular mucin. As the latter authors showed that the sialic acid content strongly depends on the age of the animals, we always used adult mice of the same age. The hexosamine analysis showed that galactosamine as well as glucosamine are present. The ratios for the sublingual and the submandibular mucins were respectively 4.5 and 1.1 (Table III). Besides galactose, mannose was also found in both mucins (respectively 3.9 and 7.7%). Moschera and Pigman [21] suggested for the rat sublingual mucin, that two different oligosaccharide chains are present. A similar structure may be present in the mouse mucins. This is especially suggestive for the sublingual mucin which contains sialic acid, galactose and galactosamine in a molar ratio of 1.00 : 1.08 : 1.03 and glucosamine and mannose in a molar ratio of 0.23 : 0.26 (Table III). Fucose and sulfate are minor components. It seems that only a small part of sugars in the carbohydrate chains carry sulfate groups. If, for example, the sulfate groups are bound to galactose, only about one out of t w e n t y galactose molecules would be sulfated. Our results are not in accordance with the histological findings for the mouse, described in the literature [2]. However, in submandibular mucins from other species sulfate has been found after incorporation of 3sSO4 [24,25] or has been detected by histochemical methods [23]. Preliminary experiments in our laboratory on the in vivo incorporation of 3sSO4 showed that in the sublingual gland about 1.4% of the radioactive sulfate is incorporated in the mucin fraction. For the submandibular gland this figure is much lower (0.07%). This may be partly due to the ten-fold lower mucin level in the submandibular gland as compared with the sublingual gland. The fact t h a t m a n y investigators are unable to detect sulfomucins in the salivary glands by histological methods, may be caused by the easy extractability of these c o m p o u n d s during the fixation [26]. The amino acid composition of the mouse sublingual mucin resembles that of the rat sublingual mucin isolated by Moschera and Pigrnan [21] in its high

440

content of Ser (20.4 vs 18.1%) and Thr (21.2 vs. 22.3%). On the other hand there is no similarity between the rat submandibular mucin described by Keryer et al. [22] and the mouse submandibular mucin, isolated by us. From the results, it can be concluded, that each gland produces its own specific mucin. The quantity of mucin, e.g sialic acid present in the glands is clearly related to the number of mucous cells [ 8 ] . Therefore the quantity of mucin or sialic acid secreted under different conditions may be used as a measure for the secretory activity of the mucous acini. Work is in progress to correlate biochemical and immunohistological findings on induction of the secretory process by sympathetic or parasympathetic stimuli. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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