The isolation and partial characterization of the principal glycoprotein from human mixed saliva

Arch8 oral Biol. Vol. 16, pp. 287-303. 1971. Pergamon Press. Printed in Great Britain.

THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE PRINCIPAL GLYCOPROTEIN FROM HUMAN MIXED SALIVA J. SCHRAGERand M. D. G. OATES The Group Laboratory,

Royal Albert Edward Infirmary, Wigan, Lancashire, England

Summary-Fifty-six individual whole salivas were fractionated on Bio-gel P150. The carbohydrate and amino acid composition of the non-retarded fraction of each was determined. The data obtained suggested that the non-retarded fraction was composed of a population of macromolecules which showed remarkable similarity in composition but were polydisperse with respect to end-groups. The results also suggested that material isolated from the non-retarded fraction was the principal glycoprotein of whole saliva as it contained 70-100 per cent of the galactosamine and 59-90 per cent of the glucosamine content put on the column. The amino acid composition of all non-retarded fractions was similar. The carbohydrate composition showed a basic structure common to all macromolecules isolated. Superimposed on the basic structure were additional sugar residues. These residues divided the macromolecules into distinctive groups. They also appeared to determine the blood group specificity as each group showed a distinctive blood group activity. All isolated macromolecules were sulphated. The basic homogeneity of the glycoproteins composing the non-retarded fraction was supported by ultracentrifugation studies. INTRODUCTION GAS-LIQUID chromatography and gel filtration have been used in this laboratory to investigate the carbohydrate-containing fractions of gastric secretion (SCHRAGER and OATES, 1968; SCHRAGER and OATES,1970). The investigation was extended to include individual whole salivas. ERICSON(1967) fractionated whole saliva on Bio-gel PI50 and isolated a nonretarded peak which contained 60-90 per cent of the hexosamine content put on the column. R~~LLAand JONSEN(1968) eluted mixed sublingual-submaxillary saliva on Bio-gel P300 and obtained a non-retarded fraction which contained all the initial blood group activity. The aim of our project was to fractionate individual whole salivas on Bio-gel PI50 and to isolate and identify, on as nearly a quantitative basis as possible, the carbohydrate-containing fractions. Preliminary studies using the methods of the investigation described in this paper have shown that the non-retarded fraction eluted at the void volume contained by far the largest portion of the carbohydrate content put on the column. The non-retarded fraction was selected as the subject matter of this study. The presence of immunoglobulins in the saliva has been reported and the literature on the subject reviewed recently (SCHULTZE and HEREMANS, 1966). These plasma 287

J. SCHRAGERand M. D. G. OATES

288

glycoproteins contain carbohydrate components (SCHULTZE and HEREMANS, 1966). The immunoglobulins IgG, IgA, IgM and a-acid glycoprotein were quantitatively determined by immuno-electrophoresis. In the current study, the carbohydrate composition of a purified sample of IgG, IgA, IgM and a-acid glycoprotein and of plasma components I, II, III, IV and V, prepared by a modification of the Cohn ethanol procedure, from “time expired” pooled human plasma were determined. It was thought that the data provided would indicate approximately the share these glycoproteins contribute to the total carbohydrate-protein complexes of whole saliva and may provide features which would distinguish these substances from the salivary glycoproteins. MATERIALS

AND

METHODS

Salivary secretion

Unstimulatedwhole salivawas collected before breakfast into an ice-chilledglasstube containing 0.5-l ml of chloroform as an antimicrobialagent. The averagesamplewas 20-30 ml. The collected specimenwas made up to O-1M withN-acetyl cysteine, pH 7.5, and incubated at 37°C for 16 hr. This proved a satisfactory method to homogenize the saliva and reduce its viscosity. The homogenized saliva was centrifuged and the deposit, consisting mainly of cells and cellular debris, as was shown by stained films, was discarded. The supernatant was further incubated with pepsin for 48 hr at pH l-5 (pepsin: substrate-l :20). An adequate quantity, 5 ml, was taken from each supematant to determine the carbohydrate composition and the sulphate and sialic acid contents. The remaining portion was eluted on Bio-gel P150 and each eluted peak was investigated. The carbohydrate and amino acid composition of the non-retarded fraction was determined. A few samples were eluted immediately after treatment with N-a&y1 cysteine in order to retain the activity of amylase. The procedure for processing the saliva is outlined in Table 1.

TABLE 1. FRACTIONATIONOF WHOLESALIVAAND SEPARATION OF TIE PRINCIPALGLY~OPROTEIN

Saliva Incubated

with 0.1 M N-acetyl cysteine for 16 hr at 37°C. Centrifugation

I

I

Deposit (Discarded)

Supernatant

I Incubated with pepsin for 48 hr at 37°C at pH 1.5

I Determination of carbohydrate, sulphate and sialic acid (3 ml)

I Elution on P150

Non-retarded fraction. Determination carbohydrate, amino acid, sulphate and sialic acid.

of

ISOLATION AND PARTIAL CHARACTERIZATION OF PRINCIPAL GLYCOPROTEIN

289

Bio-gel P150 Polyacrylamide gel was found to be superior to Sephadex for this investigation because it does not “bleed” glucose and is not attacked by bacteria. Geljiltrationprocedures andpreparation of the column. Dry beads were suspended in distilled water and allowed to swell for 5 days. The fine particles were removed by several decantations. The gel was then equilibrated with 0.05 M NaCl for 2 days. The column was designed to eliminate any large mixing volume. Two rubber bungs were selected which fitted tightly into the lower end of the glass column to be used. A bore (l-l mm dia.) was drilled in the centre of each bung to take a polythene needle. A small collecting hollow was prepared on the upper surface of the lower bung to channel the liquid into the capillary COMecthIg tube. A nylon sheet was stretched between the two bungs which were inserted into the lower end of the column. Glass beads were layered (5 cm) on the top of the upper bung. The glass column was placed in a vertical position, the lower end clamped and the column tilled with de-aerated 0.05 M NaCl solution. The suspension of the gel was de-aerated under reduced pressure before use. A plastic funnel was fitted tightly into the glass column and the slurry was passed through the funnel. The slurry in the funnel was stirred continuously to maintain an even distribution of gel. The addition of gel was continued until a required bed height was obtained and then the reservoir, fIlled with 0.05 M NaCl, was connected to the top of the column. The sample was not added until a constant column height and a flow rate of 15-20 ml per hr was obtained. To eliminate “wall effect”, the column was treated with 2 per cent (v/v) dichlorodimethylsila in benzene. This solution was heated to approximately 6O”C, poured into the clean column, allowed to stand for several minutes, the solution decanted and the benzene evaporated in a drying oven. The entire procedure was then repeated after which the column could be used for several months without further treatment.

Carbohydrate analysis. Carbohydrate analysis was carried out using the method of gas-liquid chromatography (SCHRAGER and OATES,1968) adapted in this laboratory with the following modifications. The Deacidite G resin was regenerated and then autoclaved for 16 hr at 20 lb pressure. (126°C) regenerated again and re-autoclaved to eliminate the strongly basic groups usually present in the resin. In order to obtain maximum recovery of each carbohydrate component for every specimen investigated, the range of the hydrolytic conditions was extended and each sample was hydrolyzed ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 16 hr, all at 100°C. The analytical methods for sialic acid, hexose and pepsin have been described (SCHRAGER, 1964; O~~esand SCHRAGER, ~~~~;SCHRAGER andO~-rss,1968). &&hate determination with sulphonazo III Sulphate determination with sulphonazo III was used. BUD&UNKS$and VRZALOVA,(1965) have described the reaction of sulphonazo III with barium, giving a blue colour. The precipitation of barium with sulphate reduces the colour proportionately. It has proved to be a very sensitive method. It was possible to estimate both the free and bound sulphate as follows: Free sulphate. One ml of saliva and 1 ml of 0.5 N HCl were added to a centrifuge tube followed by 5 ml acetone and 1 ml BaCL. The volume was made up to 10 ml with distilled water. The IjaSGI was allowed to precipitate overnight and then centrifuged at 4500 rpm for 15 min. An aliquot of the supernatant was withdrawn and sulphonazo III added. With the amounts of sulphate and reagents given above, a 7 ml aliquot has been used, to which was added 0.5 ml of 0.0245% sulphonaxo III solution. The colour was measured at 642 w in 1 cm cells. Total sulphate Hydrolysis. One ml of saliva was hydrolysed for 72 hr in 0.5 N HCl at 100°C. Preliminary expel+ ments have shown that under these conditions all the bound sulphate is freed, The bound sulphate value was obtained by subtracting the free from the total sulphate. Amino acid analysis The non-retarded fraction was incubated with pepsin for 48 hr and re-chromatographed reduce the impurities to a minimum. Portions were hydrolysed in 4 N HCl at 100°C for A.O.B.16/3--o

tw&

to

18 hr at a

290

J. SCHRAGER and M. D. G. OATES

concentration of approximately 3 mg/ml. After hydrolysis, the solutions were evaporated to dryness in a vacuum desiccator. The dried residue was dissolved in O-1 N HCl and amino acid analysis was carried out with the Technicon Auto Analyser. Hexose determination Total hexose were determined

by the method of WINZLER (1955), using orcinol HzS04. reagent.

Amylase determination The amylase activity was determined

by the method described by ICINGand WOOTTON

(1959).

Ultracentrifugation A Spinco model E analytical ultracentrifuge with a Schlieren optical system was used. The constant temperature control was set at 20°C and the maximum speed was 59,780 rpm. The examinations were kindly performed by Miss A. Seaston, Department of Biochemistry, University of Manchester, Manchester. Infrared anulysis Infrared spectra were measured with a Perkin Elmer model 421 spectrophotometer on RBr pressings. The analyses were carried out in the Department of Polymer Chemistry, University of Bradford, Bradford by the late Professor Moore.

The types of gamma globulins and a-acid glycoprotein present were measured by a two-dimensional gel-diffusion technique. The test fluids diffuse from the well into an agar gel containing the anti-serum specific for the component to be measured. The concentration of test globulins and a-acid glycoprotein are proportional to the diameter of the ring of immune precipitate formed and were estimated by reference to a standard curve relating ring diameters to known concentrations of purified immunoglobulins and a-acid glycoprotein (TOMAS and ZIGELBAUM, 1963). The immunoglobulins, IgG, IgA and IgM were a gift from Dr. Smith of the National Blood Transfusion Service, Manchester. The a-acid glycoprotein was a gift from Hoechst Pharmaceuticals Ltd., Brentford, Middlesex and the plasma fractions I, II, III, IV and V were prepared by Dr. Ellis, Blood Products Laboratory, Lister Institute of Preventive Medicine, London. Agglutinationinhibition. The method used was that described by BOATMANand DODD (1966). RESULTS The carbohydrate

composition

of the non-retarded

fractions

Gel chromatography resolved individual whole salivas which have been incubated with pepsin into two well-separated peaks: (1) A non-retarded peak eluted at the void volume (Fig. 1). It showed a symmetrical elution proHe of totally excluded material and contained 59-90 per cent of the glucosamine and 70-100 per cent of the galactosamine put on the column. (2) The second fraction consisted mainly of polypeptides and the remaining carbohydrate content. Untreated individual whole salivas provided three fractions when eluted on Bio-gel P150 (Fig. 2) : (1) A non-retarded peak. (2) A second peak containing mainly amylase. (3) A third peak consisting of polypeptides. The carbohydrate content not eluted in the non-retarded fraction was unevenly divided between fractions two and three.

ISOLATION AND PARTIAL CHARACTERIZATION OF PRINCIPAL GLYCOPROTEIN

Elution

of

Pepsin-digested

70 65

o

Protein

l

Hexose

A

60

solivo

Blood

group

on

bio-gel

150

activity

55 50 45 40 35 30 25 20 I5 IO 5 0 Fraction

FIG.

1. Elution pattern from Bio-gel P150 Elution carried out with O-05 M sodium chloride. Fractions of 15 ml were collected. Elution rate 15-20 ml per hour.

7ccO

c _

6000

t

Elution

8000 75 70

numbers

of

saliva

- nexose II_

o Protein

65 t

bio-gel

150

x Amylase

Fraction

FIG.

on X d: x’ ;

numbers

2. Elution pattern from Bio-gel P150. Elution carried out with 0.05 M sodium chloride. Fractions of 15 ml were collected. Elution rate 15 to 20 ml per hour.

291

292

J. SCHRACXX and M. D. G. OATES

The glycoproteins composing the non-retarded fraction are resistant to pepsin. The immunoglobulins are known to be fragmented by this proteolytic enzyme (PORTER, 1967). Current investigation of the parotid secretion in this laboratory has shown that the glycoprotein of the parotid secretion is sensitive to pepsin. The sensitivity of the immunoglobulins and the parotid glycoprotein to pepsin has facilitated the isolation of a tolerably pure glycoprotein from whole saliva. The fragmented immunoglobulins and the parotid glycoproteins were retarded and separated from the principal salivary glycoprotein which was eluted in the non-retarded fraction. All non-retarded fractions investigated contained galactose, fucose, N-acetyl glucosamine, N-acetyl galactosamine, sulphate and sialic acid. Preliminary studies have shown that the experimental error is within 5 per cent. The data also show that the variation in the experimental results is well within these limits. The quantitative relationships of the carbohydrate components are summarized in Table 2. Galactose, TABLE2.

CARJIOIWDRATE COMPONENTS OF THE NON-RETARDED FRACTION,lIiEPRINCIPAL SALIVARY GLYCOPROTEXN,OF ELUTFD WHOLE SALIVA. ?-HE RESULTS ARE EXPRESSED AS MEANS f S.D. WITH THE NUMBEROFINDNIDUALSPECIMENSINVESTIGATEDMPARENTHESIS.RA~O~FCARBOHYDRATECOMWNENT TOD-GLUCGSAhtlNE(= 3.0)

Group 1 Blood group specificity non-secretor Galactose Glucosamine Galactosamine Fucose Sulphate

Galactose Glucosamine Galactosamine Fucose Sulphate

3.90 f ::!z f 1.50 f 1.50 f

Group 2 Blood group specificity H-secretor

0.10 (7)

3.90 3.00 0.97 2.43 1.91

0.01 (7) 0.90 (7) 1.10 (5)

Group 3 Blood group specificity A & H secretor 3.90 f 0.12 (19) 3.00 1.64 f 0.20 (19) 2.73 f 0.22 (19) 2.18 f0.99 ( 5)

f 0.093 (28) f 0.042 (28) f O-32 (28) &O-94( 9)

Group 4 Blood group specificity B & H secretor 4.50 f 0.50 (2) 3.00 1:: f 0.16 8; 0.67 (1)

fucose and the two hexosamines showed distinctive quantitative relationships which divided the salivary samples into groups with respect to these quantitative differences (Table 2). The basic structure common to all salivary glycoproteins was found to be remarkably constant. It showed the following quantitative relationships: galactose : glucosamine : galactosamine 4 3 1 The molar relationships were consistently whole number ratios. Superimposed on the basic structure were additional sugar residues. These divided the salivary glyco-

ISOLATION AND PARTIAL CHARACTERIZATION OF PRINCIPAL GLYCOPROTEIN

293

proteins into distinctive groups and endowed each group with a distinctive blood group activity. The characteristic feature of the glycoproteins of Group 1 was their lower fucose content. They showed neither A, H nor B blood group specificity. The glycoproteins of Group 2 had a higher content of fucose. This was associated with blood group specificity H. It was detected in all specimens of this group. The non-retarded fractions of Group 3 has an additional galactosamine residue and the macromolecules of this group possessed blood group specificity A. An additional galactose differentiated the glycoproteins of Group 4 and these macromolecules showed blood group specificity B. These findings are in agreement with the results of Morgan and Lloyd and their collaborators (MORGANand WATKINS,1969; LLOYD, KABAT and LICERIO,1968) in their study of the glycoproteins of pseudomucinous cysts. The analytical data suggested that not all carbohydrate side chains of the glycoprotein terminated with the characteristic sugar determinant. The terminal galactosamine of Group 3, the terminal galactose of Group 4 and fucose of Groups 1 and 2 showed restricted variations. This variation of the determining sugar residues was also found in glycoproteins of pseudomucinous cysts (LLOYDet al., 1968). The division of salivary secretions with respect to blood group specificity corresponded to the division of the non-retarded fraction on the basis of the quantitative relationships between the carbohydrate components. It was thus possible to correlate and identify in chemical terms the blood group specificity of all the salivary glycoproteins investigated. The carbohydrate content of IgG, IgA, IgM and a-acid glycoprotein

The carbohydrate components of IgG, IgA, IgM and a-acid glycoprotein, isolated from plasma, were determined and the results are summarized in Table 3. It will be noticed that there are marked differences between the carbohydrate composition of the plasma glycoproteins and that of the principal salivary glycoprotein (Table 2). They differ quantitatively and qualitatively. The salivary glycoprotein contains galactosamine but no mannose whereas the plasma glycoproteins show a considerable amount of mannose but no galactosamine. The amount of mannose found in the non-retarded fraction is a sensitive indicator of the degree of contamination with plasma glycoproteins. Recovery

The recoveries of the carbohydrate content put on the column are given in Table 4. The data showed that the non-retarded fractions contained between 70-100 per cent of the galactosamine and 59-90 per cent of the glucosamine eluted from the column. The recovery rate of galactosamine was higher than that of glucosamine. This is explained by the fact that a portion of the glucosamine of the salivary secretion is contributed by the plasma and parotid glycoproteins. The plasma glycoproteins are digested by pepsin and retarded by the Bio-gel and so are not included in the nonretarded peak (PORTER,1967). Current studies in this laboratory show that parotid glycoprotein is also digested and fragmented by pepsin. The hexosamine content in

o-&lactose D-Glucosamine D-Galactosamine GFUcOSe D-Mannose Sialic acid

l-00 2.30 Trace 0.37 0.70

Serum

;:g Trace 0.66

l-00 2.00 Trace 0.50

140 140 0.20 1.00

OF THE COHN

1.00 2.00 0.50 1.00 1.33

a-Acid glycoprotein

BY A MODIFICATION

ImmUnoglobulin IgG IgM IgA

WERE PREPARED

l-00 0.80 Trace Trace 0.60

I

JTHANOL

1.00 1.20 Trace Trace 1.00

II

1.00 I.30 Trace Trace 0.60

Plasma fractions III

PROCEDURE

1,OO l-00 Trace 0.081 1.00

IV

1.00 0.66 Trace 1.00 0.64

V

TABLE3. CARBOHYDRATe COMPONENTS OF SERUM,IMMUNoGLoBULIN, a-ACIDGLYCOPROTEIN ANDPLASMA FRACTIONS. THE PLASMA FRACTIONS

9

g P

b P

;

;

%

!

rA

ISOLATIONAND

TABLE

4.

RETARDED

AMINO such4R RECOVERY. THE FRACTION

Glucosamine put on column 31.20 6.80 8.64 10.32 24.00 22.00 13.68 7.60 92.70 21.30

PARTIAL CHARACTERIZATION

OF GLUCOSAMINE

Galactosamine put on column 8.32 I.70 2.28 3.36 8.32 6.86 4.46 3.10 34.25 5.78

OF PRINCIPAL GLYCOPROTEIN

RECOVERY

(mmol)

AND GALACTOSAMINE COLUMN

m

295

THE NON-

PUT ON

THE

Non-retarded fraction Glucosamine Galactosamine 18.44 4.32 5.80 6.82 19.80 14.10 IO-56 6.90 71.68 14.04

(59-l “/,) (63.5%) (67-l %) (66-l %) (82%) (63%) (77 %) (90%) (77 %) (65%)

7-02 1.40 1.70 2-30 7.02 4.74 3-60 3.10 27.52 5.40

(84.4%) (82.4%) (74.6%) (70%) (84%) (70%) (80%) (100%) (80 %) (94%)

these substances consists mainly of glucosamine (Table 3). The exclusion of the plasma and parotid glycoproteins from the non-retarded peak would affect mainly its glucosamine content and hence its lower recovery in the non-retarded fraction. Bound and free sulphate and sialic acid

Five samples of saliva containing differing concentrations of sialic acid were divided into two portions, A and B. A was kept at room temperature (20°C) and B incubated at 37°C. The total and free sulphate and sialic acid were determined at given intervals. The results are summarized in Table 5. It will be noticed that there is a marked increase in sialic acid after 20 hr at 37”C, 95 per cent having been released. No significant changes were detected in sulphate at 37°C. The cleavage of sialic acid appears to be related to temperature and the time interval between aspiration and the investigation of the specimen. An increase in temperature accelerates the release. The sulphate content was not affected. No increase of free sulphate was observed at room temperature (20°C) or at 37°C after 6 days. These data demonstrate the differences between the sulphate and sialic acid linkages. While the latter type is very labile, the sulphate content appears to be stable under the conditions of the experiment. Sialic acid

The conditions of these experiments were unfavourable for the study of the sialic acid content of the non-retarded fraction. The data summarized in Table 5 showed that 39-76 per cent of the sialic acid was freed within 24 hr. The time interval between the collection of the specimen and the estimation of the sialic acid content of the eluted non-retarded fraction was usually between three and four days. The data relating to the sialic acid of the non-retarded fraction are unlikely to represent the total amount present during secretion. The free sialic acid would be retarded and not

-

4 5

0

0%: 0.13 0.12

I.

: 3

Sample

37°C

0.043 0.064

0.057 0.14 0.19

6 hr.

-

-

20°C

, Free

TABLE ~.THJZRELEASEOFSIALICACIDANDSULPHATE

-

37°C

OGO 0.064

16 hr.

7 -

0.076 -

20°C

,

Sialic acid

37°C

0.068 0.071

0.114 -

24 hr.

MSIALICACIDCONCENTRATION

0.121 0.16 0.35 O-068 0.071

Total

\

0 day 0.16 0.24 0.24 0.22 0.22

,

(mmol/l) AT 20°C AND AT 37°C ATDIFFERENTTIMEINTERVALSPROM

3 day 0.18 0.22 0.22 0.24 0.22

Free

,

6 day 0.16 0.22 0.26 0.28 0.26

,

Sulphate

0.67 0.84 0.78 0.76 0.82

\ Total

5 SAMPLESOFSALIVADIFPEIUNG

297

ISOLATIONNWPARTIALCHARAcTERIZATIONOFPRINCIPALGLYCOPROTEIN

included in the non-retarded fraction. The sialic acid content has, therefore, not been included in Table 2. The sialic acid contents of ten individual whole salivas were determined and the results are summarized in Table 6. These data include the contribution made by the immunoglobulins and u-acid glycoprotein both of which contain a considerable amount of sialic acid (Table 3). The sialic acid content of the salivary glycoproteins is currently being investigated under experimental conditions which, it is hoped, will not involve the release of sialic acid during the process of isolating the glycoprotein. TABLE 6. THE SIALIC ACID AND

Total sialic

CARBOHYDRATE COMPONENTS SALNAS

(mmol/l)

OF 10 INDIVIDUAL WHOLE

Galactose

Fucose

Mannose

Glucose

Glucosamine

Galactosamine

o-955 l-800 l-520 2.250 l-640 1.250 I.720 2.320 2.055 2.440

o-394 1.280 o-910 2.100 l-060 O-825 l-340 l-330 l-335 l-570

o-079 0.052 0.180 o-335 o-190 0.080 0.214 O-250 o-140 o-350

0.136 0.241 o-212 o-221 0.282 0.456 O-400 O-136 0.106 o-120

0.742 l-300 1.460 2.000 l-440 0.980 I.870 2.160 1.850 2.140

o-210 0.406 0.540 0.879 0.368 0.474 0.945 0.640 O-572 0.540

acid o-15 0.30 0.55 0.41 0.26 0.14 0.28 o-40 o-34 O-38

Amino acids

Amino acid analysis of twelve non-retarded fractions were determined (Table 7). They show the same characteristic composition found in other mucosubstances (ANDERSON et al., 1964; ADAMS, 1965). Threonine and serine constitute 45-50 per cent of the amino acid content. The ratio of threonine to serine was found to be approximately 2 : 1. Threonine, serine, proline, alanine and glycine make up between 75-80 per cent of the total amino acid content. Sulphate

The sulphate content appeared to be related to N-acetyl glucosamine. The sulphate/ glucosamine ratio varied between 1: 3 and 1: 1. The position and linkage of the sulphate have yet to be established. Immunoglobulins and a-acid glycoprotein

The immunoglobulins and a-acid glycoprotein were estimated in twelve individual whole salivas. The results are summarized in Table 8. Their carbohydrate contribution

Aspartic acid Threonine Serine Glutamic Acid Proline Glycine Alanine Wine Cystine Zso-leucine Leucine Tyrosiue j3-Phenylalanine Lysine Histidine

0.50 1.90 0.60 0.60 0.90 0.50

3.10 4.60 1.20 -

1 1*90 lO*OO 5.70 2.40 -

2 1.10 10.00 5.30 2.40 5.60 3.30 5.50 1.10 Trace 0.50 2.10 Trace 2.70 I.30 1.20 0.80 1.90 0.90 0.80

1.20 0,80 0.60

4 1.80 lO*Oo 5.50 2.20 5.50 2.80 4.30 1.20

O-50 1.90 -

3 1.20 10.00 5.30 2.90 1.40 5.00 Trace 3.30 Trace Trace Trace Trace

3-40 5.20 -

5 1.30 10.00 5.40 2.70 -

0.60 2.20 Trace Trace O-90 O-80

2.90 4.00 1.30 -

6 2.10 lO*OO 4.70 2.90 -

0.30 0.80 0.60 0.40 0*60 0.60

0750 10~00 4.60 1.30 4.90 2.10 4.10 0.60 1.90 0.60 0.90 l-10 0.80

3.00 4.40 0.90 -

8 1.50 10.00 5.30 2.80 -

10 2.00 lO*OO 4.90 3.00 3.40 4.60 l-60 0-60 1.70 0.90 l-30 -

9 1.70 10~00 5.40 2.30 2.30 3.80 1.40 0-60 3.60 I.50 0.80

OG 0.90

2.20 -

1

11 2.00 lO*Oo 5.90 2.30 5.80 3.90 4.60 -

0.70 2.20 o-50 1.50 0.90 O-80

3.20 5.00 -

12 1.10 10.00 5.00 2.10 -

TABLE 7. AMINO ACID COMPOSITIONS OF THE NON-RETARDED FRACTION OF 12 RJZPRESENTATIVE SALIVAS ELUTED ON BIO-GEL P150. VALUES ARE IN MOLAR PROPORTIONS (THREONINE = 10)

ISOLATION

AND

PARTIALCHARACTERIZATXONOFPRINCIPALGLYCOPROTEIN

299

TABLE 8. IMMUNOGLOBULIN AND ALPHA-ACID GLYCOPROTBIN CONTENTSOF 12 INDIVIDUALWHOLESALIVAS(~~~~~~OO~~).EACH INDIVIDUAL SALIVA WAS CONCENTRATED BY FREEZE-DRYING AND RESTORED TO l/lo OFTHE ORIGINALVOLUME

I@ 10 10 10

IgA 5.5 4.2

Approx. l-5 Approx. l-5

4.5 6.4 15.5 16.0 17.0

10.5

16.0 10.0 13.5

-

4.5

IiW 5.5 -

q

a-acidglycoprotein

1.4 1.0 -

-

to the whole salivas investigated is only small but they could affect the glucosamine/ galactosamine ratio as the hexosamine of these glycoproteins consists mainly of N-acetyl glucosamine. Their amino acid composition differs fundamentally from that of the salivary glycoproteins (PORTER, 1967). Contamination of the salivary glycoproteins with even small amounts of immunoglobulins would alter greatly the amino acid composition of the salivary glycoproteins as the latter contain only 15-30 per cent of protein whereas the immunoglobulin IgG contains 98 per cent and the immunoglobulin IgA contains 90 per cent of protein (PORTER, 1967). Infrared analysis

The 1240 (S-O present.

stretching) band, characteristic

of sulphate, was found to be

Ultracentrifugation

Ultracentrifugation studies revealed a uniform within the limits of this method (Fig. 3).

peak suggesting homogeneity

DISCUSSION

This investigation aimed at isolating the carbohydrate-containing fractions found in whole saliva and determining their composition. Gel chromatography proved to be a simple and efficient method for fractionating salivary secretions and gas-liquid chromatography was demonstrated as a powerful separating technique which would rapidly provide reproducible data on complex sugar mixtures. Fifty-six individual samples of whole saliva were eluted on Bio-gel P150 into wellseparated fractions. The non-retarded fractions contained 68-95 per cent of the carbohydrate content added to the column. The carbohydrate components of all

300

J. SCHRAGER and M. D. G. OATES

non-retarded fractions investigated showed a basic common structure of constant composition. Additional sugar residues divided the glycoprotein into distinctive groups, each group having a characteristic sugar residue correlated with a specific blood group activity. The analytical data thus uncovered a chemical basis for the blood group specificity of salivary glycoproteins. The protein moiety of salivary glycoproteins accounted for only a minor portion of the entire molecule (15-30 per cent) (Table 9) and any attempt at its characterization, therefore, requires the complete removal of non-covalently bound proteins and glycoproteins. The macromolecules would be expected to absorb small polypeptides and this would increase the difficulty of purification. Despite these difficulties, the amino acid composition of the protein core was found to be approximately constant in all salivary glycoproteins (Table 7). The protein moiety showed characteristic features found in other mucosubstances. Threonine and serine made up 45-50 per cent of the total ammo acid content, but only traces of cysteine and no methionine were found. The two hydroxy amino acids, together with proline and alanine, were the major amino acids constituting 70-80 per cent of the protein core. The constancy of composition, especially as it is expressed in the basic common structure of the carbohydrate side-chain, the approximate constancy of the amino acid composition and the ultracentrifugation studies suggest that the isolated nonretarded fraction consists of macromolecules which are all of the same overall chemical structure but show polydispersity with respect to their end group and charge. The data imply that they consist of a protein core to which are attached numerous carbohydrate side chains. Current investigations in this laboratory suggest that the side chains are linked to threonine and serine, galactosamine forming the link; thus statistically half the amino acid residues of the protein core could carry side chains. The data also suggest that their macromolecules constitute the principal glycoprotein of saliva as it contains 59-90 per cent of the glucosamine and 70-100 per cent of the galactosamine put on the column. The results of this investigation provided distinguishing features which characterize the principal glycoprotein found in saliva: (1) The quantitative relationships between the carbohydrate components. The basic structure showing: galactose 4

:

glucosamine 3

:

galactosamine 1

(2) The presence of galactosamine and absence of mannose. (3) Blood group specificity. (4) An unusual but characteristic amino acid composition. (5) The high carbohydrate content of the glycoprotein (70-88 per cent) (Table 9). The results of this investigation may be reconciled with those of other workers in this field (MANDEL and ELLISON, 1961; FISCHER,WYSHAK and WEISBERGER, 1962; WEISS and HABER, 1964) who have maintained that saliva contains a number of

Total sugars

7.335 l-258 l-818 2.852 2.298 l-353 4.040

2.183 O-698 l-206 1.334

l-010 l-375

3.095

Total amino acids

1.256 0.167 O-382 O-810 o-264 O-324 o-941

o-492 O-286 0.494 0-272

O-386 0.257

0.802

TABLE 9. THE TOTAL

AMINO

l-100

o-330 o-375

72 84 79

o-579 o-229 O-648 O-380

2-100 O-358 O-552 0.760 o-570 o-395 l-120

0.340

0.253 0.091 O-216 o-130 O-128 0.125

O-142 0.430

0.915 0.172 o-204 o”:E

Amino sugars Galactosamine Glucosamine

THREONINEAND SERINSCQNTENT 14 SPECIMENS OFWHOLESALIVA

70 70 83

;: 89 86 ::

86 88

0%of sugars in glyccprotein

ACID, CARBOHYDRATE AND FRACTION OF

o-260

o-149 o-074 O-158 0.080 0.092 O-083

0.396 O-046 o-120 o-249 O-069 0.093 O-231

Threonine

o-121

0.074 0.037 O-085 O-046 o-049 O-044

0.207 O-023 O-065 0.110 o-041 0.045 0.125

Serine

(mmol/l.) OF THE ELUTED

H

AandH Non-sec. H H H H

AandH AandH AandH AandH AaudH AandH AandH

group

specificity

Blood

NON-RETARDED

? ;: 8 Fz

2 5 g F

P

EI 8

P

$

i

k

8 2:

p

302

J. SCHRAGERand M. D. G. OATES

carbohydrate-protein complexes. Electrophoresis and ion-exchange chromatography were almost the only methods used by these workers to fractionate salivary secretion. The colloids which constitute salivary mucus secretion do not lend themselves easily to electrophoresis. Highly charged polymers such as these produce viscous solutions. The concentration at which the initial band must be applied to the paper is so high that the glycoprotein, itself, is a gel-like structure forming a viscous elastic network not easily and uniformly acted upon by electrophoretic forces. This investigation showed that the salivary glycoproteins are polydisperse with respect to sulphate and sialic acid. Variations in the proportion of the charged end groups attached to these macro-molecules result in differing degrees of electrophoretic mobility and of absorption of ion exchange resins. The variations in the number of sulphate and sialic acid end groups is reflected in the stepwise changes in electrophoretic mobility. It is, therefore, suggested that many of the multiple bands or fractions obtained by these separation procedures arise from variations in these two groups attached to the main basic macromolecular component rather than from major differences in its “backbone” structure. The evidence presented in this paper is consistent with a concept that the isolated glycoprotein is a compound of a basic homogeneous composition and structure but polydisperse with respect to reactive end groups and charge. R&sum&La salive integrale de 56 individus a et& fractionnQ sur Bio-gel P150. La composition des hydrates de carbone et des amino-acides de la fraction non-retard&. de chaque, a et& dtterminee. Les dates obtenues suggerent que la fraction non-retard& dtait composee dune population de macromolecules qui montrait une remarquable simiIaritC dans la composition, mais une polydispersion en ce qui concerne les groupements finals. Les r&.ultats suggeraient aussi que le materiel isole de la fraction non-retarded etait la glucoproteine principale de la salive integrale, &ant donne qu’elle contenait 70100 pour cent de galactosamine et 59-90 pour cent du contenu de glucosamine mis dans la colonne. La composition des amino-acides de toutes les fractions non-retard& etait similaire. La composition des hydrates de carbone montrait une structure de base commune a toutes les macromolecules isolees. Superposes sur la structure de base il y avait des residus additionnels de sucre. Ces residus divisaient les macromolecules dans des groupes distinctifs. 11s apparaissaient determiner aussi la sp6citicite des groupes sanguins, &ant donne que chaque groupe montrait une activite distinctive des groupes sanguins. Toutes les macromolecules isol& etaient sulfatees. L’homogeneitt de base des glucoproteines composant la fraction non-retard&e 6tait appuyee par des etudes d’ultracentri-fugation. Zusammenfassung-Sechsunfiinfzig individuelle Gesamtspeichelproben wurden auf B&Gel P150 fraktioniert. Es wurde die Kohlenhydratund Aminosaurezusammensetzung der nicht retardierten Fraktion jeder einzelnen Probe festgestellt. Aus den so gewonnenen Unterlagen ist anzunehmen, dass die nicht-retardierten Fraktionen aus einer Population von Makromolektilen zusammengesetzt sind, welche einander in Bezug auf Zusammensetzung ausserordentlich %hneln, jedoch in Bezug auf Endgruppen polyzerstreut waren. Die Resultate fiihrten such zur Annahrne, dass das von den nichtretardierten Fraktionen isolierte Material der Haupt-Glykoproteinbestandteil des Gesamtspeichels war, da es 70-100 Prozent des Galaktosamins und 59-90 Prozent des auf der Saule erscheinenden Glukosamininhaltes umfasste. Ahnlich war such die Aminoslurezusammensetzung aller nicht-retardierten Fraktionen. Die Kohlenhydratzusammensetzung zeigte dieselbe Grundstruktur wie

ISOLATION AND PARTIAL CHARACTERIZATION OF PRINCIPAL. GLYCOPROTEIN

303

bie allen isolierten Makromolektilen. Die Grundstruktur war von zusiitzlichen Zuckerresidien tiberlagert. Diese Residien teilten die Makromolektilen in deutlich unterscheidbare Gruppen ein. Sie schienen such die spezifischen Blutgruppen festzusetzen, da jede Gruppe eine charakteristische Blutgruppenaktivitat aufwies. Alle isolierten Makromolektilen wurden sulfatiert. Die Annahme der grundsatzlichen Homogenitlt der Glykoproteins, aus welchen sich die nicht-retardierten Fraktionen zusammensetzen, wurde durch ultrazentrifugale Studien bekraftigt. REFERENCES ADAMS,J. B. 1965. Studies on the mucin derived from human colloid breast carcinoma. Biochem. J. 94368-377. ANDERSON,B., NOBUKO, S.,SAMPSON, P., RILEY, J. G., HOFFMAN,P. and MEYER,K. 1964. Threonine and serine Linkages in mucopolysaccharides and glycoproteins. J. bioi. Chem. 239, (8). BOORMAN,K. E. and DODD, B. E. 1966. An Introduction to Blood Group Serology, Chap. 8, pp. 61-66. Churchill, London. BDDBSCNKSQ, B. and VRZALOVP;,D. 1965. Complexes of metallochromic substances. VII. Z. Analyt. Chem. 210,261-266. ERICSON, T. 1967. Adsorption to hydroxylapatite of proteins and conjugated proteins from human saliva. Caries Res. 1,52-58. FISCHER,C. J., WYSHAK,G. H. and WEISBERGER, D. 1962. The separation of salivary proteins by paper electrophoresis under various conditions. Archs oral Biol. 7,297. KING, E. J. and WOO~~ON,I. D. P. 1959. Micro-Analysis in Medical Biochemistry, Chap. 4, p. 91. J. & A. Churchill Ltd., London. LLOYD, K. O., KABAT, E. A. and LICERIO, E. 1968. Immunochemical studies on blood group. XXXVIII. Structures and activities of oligosaccharides produced by alkaline degradation of blood-group Lewis” substance. Proposed structure of the carbohydrate chains of human bloodgroup A, B, H, Lea and Leb substances. Biochem. 7, (8), 2976-2990. MANDEL,I. D. and ELLISON,S. A. 1961. Characterisation of salivary components separated by paper electrophoresis. Archs oral Biol. 3, 77. MORGAN, W. T. J. and WATK~S, W. M. 1969. Genetic and biochemical aspects of human blood group A-, B-, H-, Lea- and Leb Specificity. Br. Med. Bull. 25, 30-34. OATES, M. D. G. and SCHRAGER,J. 1967. The determination of sugars and ammo sugars in the hydrolysates of mucopolysaccharides by gas-liquid chromatography. J. Chromat. 28, 232-245, PORTER.R. R. 1967. The structure of immunoalobulins. ESSUYSBiochem. 3.1-24. R~LLA,‘G. and JONSEN,J. 1968. A glycoprotein component from human sublingual-submaxillary saliva. Caries Res. 2,306-3 16. SCHRAGER, J. 1964. Sulphated mucopolysaccharides of the gastric secretion. Nature, Land. 201. 702-704. SCHRAGER,J. and OATES,M. D. G. 1968. The carbohydrate components of hydrolysates of gastric secretion and extracts from mucous glands of the gastric body mucosa and antrum. Biochem. J. 106,523-529. SCHRAGER,J. and OATES,M. D. G. 1971. Further observations on the principal glycoprotein of the gastric secretion. Digestion (in press). SCHUL~ZE,H. E. and HERE%iANS, J. F. 1966. Molecular Biology of Human Proteins, Chap. 5, pp. 773815. TOMASI,T. B. JR. and ZIGELBAUM,S. D. 1963. The selective occurrence of gamma-1A globulins in certain body fluids. J. Clin. Invest. 42,1552. WEISS,I. T. and HABER,N. 1964. Evaluations of paper electrophoresis. J. dent. Res. 43,652. WINZLER, R. J. 1955. Determination of serum glycoproteins. Meth. Biochem. Anul. 2, 279-311.

PLATE 1 OVERLEAF

ISOLATION

AND

PARTIAL

CHARACTERIZATION

OF PRINCIPAL

GLYCOPROTEIN

FIG. 3.-Ultra-centrifugation of a non-retarded fraction of eluted whole saliva on Bio-gel 150. The ultra-centrifugation conditions were 59,780 rpm. Temperature 20°C. Approximately l-5 mg/ml in 0.2 M sodium chloride.

PLATE

1

A.O.B. f.p.304