Proline-rich proteins in membranes and contents of monkey (Macaca irus and Cercopithecus aethiops) parotid zymogen granules

,qrch.~ oral Bhd. Vo|, 21, pp. 379 IO 383, Perl~:tmorJ Presn 19715. Printed in Greatt Britain,

PROLINE-RICH PROTEINS CONTENTS OF MONKEY

IN MEMBRANES

AND (MACACA IRUS A N D CERCOPITHECUS AETHIOPS) P A R O T I D ZYMOGEN GRANULES

P. A R N E B E R G * , K. HELGELAND and T. T J ( ) R N H O M Department of Microbiology, Dental Faculty, University of Oslo, and The Agricultural College of Norway, As, Norway S u m m a r y - - M e m b r a n e proteins and extractable proteins of the parotid zymogen granules were investigated as a possible source of a basic secretory glycoprotein rich in proline, glycine and glutamic acid. Isolated zymogen granules from glands of m o n k e y s were extracted. T h e sedimentable material after extraction appeared to consist mainly o f m e m b r a n e s when assessed by electron microscopy. The extracted proteins, assumed to represent the n o n - m e m b r a n e part of zymogen granules, were fractionatcd by isoelectric focusing. The proline-rich basic glycoproteins, like regular secretory proteins, were present in the n o n - m e m b r a n e part of zymogel~ granules. The results do not support the concept of zymogen granule membranes as precursors of secretory glycoproteins. INTRODUCTION

In parotid saliva in man, glycoproteins of high isoelectric points account for a b o u t one half of the total protein (Arneberg, 1972). These basic glycoproteins occur in several molecular forms. In all of them, proline, glycine and glutamie acid occur at a ratio of 2:1:1, and account for 70-80 per cent of total a m i n o acid residues (Levine, Eltison and BahL 1973; Arneberg, 1974). A similar amino acid composition has been reporled for the m e m b r a n e of the zymogen granule in the rat parotid gland (Amsterdam et al., 1971). It was suggested that proteins o f this m e m b r a n e might represent precursors of secretory glycoproteins and that their glycosylation might be coupled with the secretory release from the acinar cell. The p u r p o ~ of this study was to gain further information a b o u t the intracellular origin of this m a j o r glycoprotein fraction and of their possible association with the zymogen granule membrane. The parotid glands o f m o n k e y s were selected because proteins of similar basic character are released from this gland during #~ vitro cultivation (Jacobsen, 1970). MATERIALS AND METHODS

Isolation of zymogen granules A subcellular fractionation procedure for isolation of zymogen granules from the parotid glands of Macaca irus and Cercopithecus aethiops m o n k e y s has been worked out, and the degree of purity assessed by m a r k e r enzymes and d e c t r o n microscopy (Arneberg, Dahl a n d Hars, 1975). Extraction of zymogen granules Separation of the soluble proteins from the membrane was carried out by extraction of the granule fraction in 1 or 10 m M of tris/hydrochloric acid buffer, p H 8.5, containing 0.05 m M E D T A and 0.2 * Present address: Klinikk for Konserverende Tannpleie, Odontologiklinikkene. Geitemyrsveien 71. Oslo 4, Norway. 379

ttg/ml N,N'-diphenyl p-phenylenediamine (Amsterdam et at., 1971). T w o different extraction procedures were used.

Extraction by dialysis (Amsterdam et al., 1971). Z y m o g e n granules (7-14 ml), as isolated in a gradient of strongly hypertonic sucrose (Ameberg et al., 1975), were dialyzed for 16 br at 4°C against 300 vol of the ]0 m M alkaline barfer described above. Dialyzing tubes 1 crn in diameter (No.4465/Az A. T h o m a s C o . ) w e r e used. The dialyzed material was combined with the buffer used for washing the inside of the bag and centrifuged at 20,000 gn~a~ for 20 mix at 4°(2, with a SS 34 rotor in a RC2B centrifuge (Sorvall Inc. Co.). The supernatant with the soluble proteins was removed by aspiration and represented the extractable (non-membrane) fraction. The sediment was suspended in the I m M buffer and recentrifuged. The second supernatant, containing very low levels of p r o tein and n o amylase activity, was discarded and the sediment used. as the crude m e m b r a n e fraction. Extraction by filtration T h e zymogen granules were sedimented, resuspended in 10 ml of the 10 m M alkaline buffer and applied to a 15-rrd ultrafiltrafion cell (Amicon type) equipped with a 0.22 /an eelttalose filter (Millipore Co.). Gentle stirring and a pressure of 3 aim. N2 were applied during filtration. Before all liquid had entered the filter, 10ml of the 1 0 r a m buffer and subsequently 10ml o f the 1 m M buffer were used for washing. The combined filtrate gave no sediment u p o n high-speed centrifugation a n d r e p r e s e n t e d the extractable (nonmembrane) fraction. T h e crude m e m b r a n e fraction was recovered from t h e filter by reversal o f the flow. T h e procedure was completed in 2 hr. Purification of membranes T h e crude m e m b r a n e s were suspended in 1.0 b t sucrose solution (0.75 ml) in non-wetting tubes a n d overlaid by 0.3 M s u c r o s e according t o Meldolesi, Jamieson a n d Palade (1971). A Beckman Ti 56 rotor

380

P.

A~twber& K. l lclgcland amd T. "l++ii+,rnh,,,ml

was used for ccntrifl~gation at 150.0tl}q,~,, for one hour at 4':'C. The purified membrane fraction w a s obtained as a whitish lxmd in the interptm~. Is~:~gcctric fi~<'usi~l Atx'~ut 6 mg o f solubilized (non-membrane} z 3 ~ o g e n granule protein, as obtained by the dialysis proc~x~are, was fractionated in a 1 lt~.ml column (LKB 8101). T h e lower d e c t r o d e was used as the cxJthode+ Carrier ampholyte {Ampholine ~, LKB) of the pH ranges 3-10 (l.5g) and 7-+10 (0.5g) formed the pH gradient, and a 40-0 per cent sucrose gradient was used to stabilize the zones. Focusing was carried out at 3t30 V for 4 hr followed by 5~30 V for 48 hr. The current decreased f r o m 4 toO.5 m M during the run. T h e proteins Of the eluted fractions wcrc separated from sucrose and ~arrier ampholytes b y gel filtration and measured a s described previously (Arneberg, 1972). Amh~o acid analysis Samples containing 0.1-:1.0 nag proteins were taken to dDmess in a rotary evapoi4ator, hydroi~,w.ed in 6 N hydrochloric acid under nitrogen, re-evaporated and dissolved in 0.05 M acid. Subsequently, the hydrolysate was purified on a cation exchange c o l u m n (Dowex 50 W 100-200 mesh} and derivatives were prepared for gas-chromatographic quantitation of the amino acids, according to the method o f Zanetta and Vincendon (1973). Electron microscopy T h e zyrnogen granules were processed as described prev!ously (Arneberg et al,, 1975). The membrane spemmen was fixed for 4 h r in 2"per cent glutaralde, hyde, buffered at p H 7.3 by 0.1 M .sodium phosphate, postfixed for one hour in 1 per cent osmium tetroxide in the same buffer arid dehydrated for e m b e d d i n g in Vestopa| W®. RESULTS

Extraction experiments Whether extraction was carried OUt by filtration o r b y dialysis,~ the remaining material of the zymogen granules ( t h e c r u d e membrane fraction) accounted for

5+10 per cent of the zymogcn gramdc frm:tion at~d less than 0.5 per cent of its amyla.~ activity {Table 1). No intact zymogen granules were detected by dec° tron micro~ropy of crude membranes, as obtained by the dialysis procedure. Remnants of the granule content were o b ~ r v e d in only a few of the membrane vesicles {Fig+ 2 a,b). Extraction by the dialysis procedure solubilized 30--70 pep cent of the zymogen granule protein and a corresponding part of the amylase activity (Table 1)+ The f r a c t i o n was assumed to repres'ent the zymogen granule content (non-membrane fraction). In comparison, more than 90 per cent of protein and amylase were recovered as non+membrane fraction when flae filtration method was employed: When crude membranes were purified by gradient cenirift~gation; a considerable loss oecurred {'Fable 1). Amino acid composition of the extractable (non-membrane)fractitm; This fraction had cssentiaUy the same amino-acid profile whether obtained by the fillration or the dialysis prc,cedure. This suggested that the incomplete recovery a f t e r dialysis did nol represent a loss of specific proteins {Table 2b). Proline, glycine and glutamic aci~t were present in similar amounts, representing about 60 per cen~ of the total amino acids. Minor differences between the two samples were observed only in phenylalanine, tyrosine and serine content. Amino acid composition of the membrane fi'action. Different amino acid profiles were found for membrane fractions isolated by different procedures (Table 2a). The dialysis procedure gave a crude membrane fraction, differing little from that of extractable (nonmembrane) fractions in amino acid profile, except for a higher content of basic amino acids and methionine and lower content o f serine {Table 2a). Membranes obtained by the dialysis procedure and further purified by gradient centrifugation were characterized by a low content of glycine, whereas the proline content was similar to the extractable (nonmembrane} fraction, This analysis w a s based on only 0.4:/,moles of derivatives of amino acids, and amino acids present in low amounts could not be reliably measured.

Table 1. Isolation of zymogen granules: a n d subsequent fractionation i n t o m e m b r a n e a n d " n o n - m e m b r a n e " fractions Specific enzyme activity* Total protein* mg Isolation Of zvmo~Un ~granules: :Parotid gland h o m o g e n a t e ZYmogen granules ExtractiOn of:zvmogen granulest: "N0n=membtane" ~fraction Crude membrane: fraction iPutbifi.ca!f6n o f c r u d e mere.branes~ Purified membranes Rest :of t!le gradient Sediment

Amylase

Cyt0chrome c oxidase

949,0 37.3

345 539

0.51

11.4 2.3

578 25

0.2 °9 0.5

<5

1.94

* Analytical procedures arid enzyme activities as defined previously (Arnesberg et at., 1975). ~ Extra&iUn by: dialySis pi'ocedures.

gt'al~C~ b, "N on- m,:mb~an¢" hactio~

a. M e m b r a n e fractk, n

C r u d e mere- fh.trilied C r u d e m e m branes membranes branes (extr~tc{extrac(extraction by l i o n by lion by Extraction Extraction dialysis) dialysis) f i i t r a ' i o n ) by d~l~,.~i~ b y lfihtrali6~ (i) 12) (3) {2) (31

pl 9 |~act~o~l (4)

p! ,~ ~r;tctio~ (4)

Ala Val

5.6 3.6

65) 4.3

g.5 5.8

4.~t 2.7

5.3 23

l.I 0.7

~t.k~) ((t.7 ~

2_~ I. t

¢6I) ~60

lie Leu

2.6 5.4

5,0 5.2

5.0 8.5

2.0 4.3

I .g 3.3

0°3

(0.01

2,4

~5.0,*

0.8

{0.0~

2,¢a

~5.5~

Tar Ser Met Phe Asp O lu Tyr Lys H is Arg

29 5.2 0.4 2.5 6.5 .1_. ~ . 0.9 5.9 0.0 3.9

4.5 8.4 (k() 2.4 9.0 J.2~7 0.5 4.0 0.0 ! .2

4.8 7.7 0.0 2.8 9.1 J4g~ 1.8 6.3 0.0 3.9

2.2 ! 1.3 0.0 3.5 7.t ~ 0.9 4.4 0.0 2.2

0.0 3.7 0.O 0.2 4.2

10.0) {4.2) (0.0l (0.01 (4.8) ( 19.0 ) (0.O) (491 (I.OI (4.4t

2.4 9.5 0.0 1.3 10.0 ~ 0.4 5.1 52 .t.4

~4A) ~S.2~ t i. 7 (SAt {13.8; {8_2 l(ZS~ (5.5i t 2.21 (6,11

2.1 ~.0 0.0 ! .9 6.8 L0._~4 1.6 5.0 0.0 1.9

~ 0.0 5.0 0.5 4.3

T h e p r o t e i n s w e r e o b t a i n e d from four s a m p l e s o f m o n k e y z~xnogen granules, ( I ) a n d (4) from Maca~x~ irus a n d (2) a n d (3) from Cercopithecus aethiops. * H u m a n p a r o t i d basic s e c r e t o r y g l y c o p r o t e i n (Levine et aL. 1973l. t M a c a c a irus p a r o t i d s e c r e t o r y a l p h a a m y l a s e ( J a c o b s e n a n d S6nju. 19711. In m e m b r a n e s o b t a i n e d by the filtration p r o c e d u r e , the c o n t e n t o f proline, glycine a n d g l u t a m i c acid was significantly lower t h a n in the e x t r a c t a b l e ( n o n - m e m b r a n e ) fraction (Table 2a.b). Fractionation o f the extractable (non-membrane) proreins and subsequent amino acid analysis. A b o u t 4 nag p r o t e i n was r e c o v e r e d after isoelectric focusing, o f which o n e lmlf f o r m e d a basic p e a k o r b a n d near p H 9 in the a l k a l i n e e n d o f the g r a d i e n t (Fig. I). An acidic p e a k n e a r p H 4 c o n t a i n e d a b o u t I m g protein. T w o small p e a k s a t p H 5 a n d 7.5 were n o t ana157.ed for a m i n o a c i d c o m p o s i t i o n . T h e p I 9 fraction had a c h a r a c t e r i s t i c anaino-acid profile in which p r o l i n e a l o n e a c c o u n t e d for 35 per 12 pH

TOTAL

PROTEI N mg

""~]~-.. ,ob¢ ........ .graclioo.

9 6 3

. . . . . 5. . . . . . . . . . 5 nnl

15

fractions

Fig. 1. Extractable (non-membrane) zymogen granule proteins, separated b y isoelectric focusing: Six mg protein extracted from Macaca irus zymogen granules by the dialysis procedure was fractionated in pH gradient 3-10. pH and protein content of fractions eluted from the isoelectric focusing column.

cent a n d , t o g e t h e r with glycine a n d g l u t a m i c acid. for a b o u t 80 p e r cent o f the t o t a l a m i n o a c i d s ( T a b l e 2c). In the p l 4 fraction, these three a m i n o a c i d s a c c o u n t e d l o t a total o f 52 p e r cent. p r o l i n e a n d glyc i n e levels b e i n g c o n s i d e r a b l y l o w e r t h a n in the pl 9 fraction (Fig. 2eL

DISCU.~SION T h e s e c r e t o r y p r o t e i n s in zy'mogen g r a n u l e s are segregated from the c y t o p l a s m b y a unit m e m b r a n e . Little is k n o w n a b o u t the b i o s y n t h e s i s a n d u l t i m a t e fate o f this m e m b r a n e . E l e c t r o n m i c r o s c o p i c o b s e r v a tions on the secreting a c i n a r c e l t o f the rat p a r o t i d g l a n d after i s o p r e n a l i n e s t i m u l a t i o n suggested that this m e m b r a n e b e c o m e s p a r t o f the luminal p l a s m a membrane during exocytosis of the granule content. It was considered" that this excess o f p l a s m a m e m b r a n e w a s s u b s e q u e n t l y r e s o r b e d a n d p o s s i b l y re-utilized ( A m s t e r d a m , O h a d a n d S c h r a m m . 19691. O n the o t h e r hand. a b i o c h e m i c a l s t u d y indicates t h a t t h e ~ m e m b r a n e p r o t e i n s a r e secreted a n d not re-utilized. b e c a u s e their a m i n o a c i d c o m p o s i t i o n is chan~cteristic o f h u m a n s e c r e t o r y g l y c o p r o t e i n s a n d b e c a u s e their t u r n o v e r r a t e in the a c i n a r cell is q u i t e high ( A m s t e r d a m et aL, 19711. T h e p r e s e n t s t u d y was c a r r i e d o u t p a r t l y t o see if the preferential l o c a l i z a t i o n o f the p r o l i n e - r i c h p r o teins in m e m b r a n e s , o b s e r v e d with r a t s o c c u r r e d also w h e n z y m o g e n g r a n u l e s from m o n k e y s Were used. T h i s was n o t f o u n d either with the original e x t r a c t i o n

3~2

I', ,'%inclx'tg, K l lelg,:hind ;:lll,i;t 1, Tjlirrihoill

pr~'txture, or wheat filtration or gradient centrifugalion was u,,;exl for membrane purification (Table 2a, b}. Our results therefore do not support a precursor role of the membrane proteins in glycoprolein secretion. A o-ucial point is the purity of the zymogen granules u ~ d Ibr extraction, as particulate contaminants tend to accumulale in-'the membrane fraction. However, a high purity of the granules u ~ d has been suplx~rted by enz~anic and morphological data (Arneberg et al., 1975). Observations on rat parotid zymogen granules have confirmed the original observations of Amsterdam ct al., (1969) that proline-rich proteins accumulate in the membrane fraction and that these proteins show a high lurnover rate similar to that of secretory proteins (Robinovitch et al,, 1975: Wallach. Kirschner and Schramm, 1975). However, these studies also showed that the proline-fich proteins could be extracted from the membranes by buffers of high tonicity or low pH, Their presence in the membrane fraction amy therefore be an artifact caused by ionic interaction during the extraction procedure. It is thus interesting that the lowest proline content in the membranes was obtained by filtration which gives the shortest exposure time of released proteins to merebrines {Table 2a). The rapid turnover ra~,e of the proline-rich proleins in rat ztanogen granule membranes may also argue against their being lrue membrane components, as experiments with pancreatic glands showed a slow turnover rate of t h e zymogen granule membrane (Meldolesi, 1974). Nevertheless, a role of the prolinerich proteins in the secretory process cannot be disregarded, as specific increases in s u c h components are effected by stimulation of secretory activity in man (Levine et al., 1973) and in the b a b o o n (Williams and Keller. 1973} as well as by isoprotencrol treatment o f rats (Fernandez-Sorensen and Carlson, 1974). These data are difficult to interpret, however. because the human parofid gland secretes-a number of proline-rich proteins, some of acidic character and mainly non-glycoproteins (Bennick and Connell, 1971; Oppenheim, H a y and Franzblau, 1971)., and some o f basic character and mainly glycoproteins (Arneberg, 1974). It is further possible that this variety o f proline-rieh proteins arises by degradation in the parotid gland of a few primary gene products (Azen, 1972; Azen a n d D e n n i s t o n , 1974). The effects o f secret o r y stimulus and isoprotenerol m a y therefore be on the r a t e of degradation just as as well as on their biosynthesis. This problem m i g h t be .studied by comparing proline-rich proteins in the secretion with precursors from different tevels o f their intracelluiar pathway in the acinar cell. In o u r study, acldic a n d basic prolinerich proteins were isolated from t h e c o n t e n t o f monkey zyrnogen granules, The basic proteins constituted t h e m a j o r part o f the zymogen granule content and had the ,same characteristic amino acid profile as a corresponding fraction i n the h u m a n secretion (Fable 2c). Acknowledgements-2-we wish": to thank ; Renate Hats, Department Of Anatomy(electr0n microscopy) for expert assistance. Dr, Liv Helgel~qd, Institute of Biochemistry,

tq~Ivcrsity of ()slo and limdly Anctt~ Mcl~om. l'ubl~hi~Ig 0i1i¢¢. 1}cain| F'aculiy for lingliistic advice. REFt~RENCt;S Amsterdam A., Ohad I. and Schramm M. 19159. Dynanfic ehat~ge.~ in the ultrastruclure of the acinar cell of the rat parotid $|and dur;,ng the secretory cycle. 2. Celt Biol, 41. 753.;,773. Amsterdam A.. Schramm M,, ()had I., Salomon Y, and S¢linger Z. 1971. Concomitarlt synthesis of membrane protein and e~portable protein of the secretory gr;mlde in rat parolid gland. J. Cell Biol. fffL 187--2(K). Arneberg P. 1972 Fractionation of parotid salivary proteins by isoelectri¢ focu:;ing in a wide ptt range. Stand. J. dent. Rcs. 80," 134-138, Arneberg P. 1974. Partial characterization of five glycopto. rein fractions secreted by the human parotid glandz. Arch,~ oral Biol. 19. 921 92S. Arneberg P., Dahl E. and liars, R. 1975. Purification of zymogen granules from monkey parofid glands. Acta path. microhiol..~cand. Sect. A. 83. 389.-394. Azen E. A. 1972. Genetic polymorphism of basic proteins from parolid saliva. Scicm'e 176, 673-674. Azen E, A. and l)enniston C. L. 1974. Genetic polymorphism of human salivary proline-rich proteins: further genetic analysis. Biochem. Genet. 12, 109-120. Bennick A. and Connell G. E. 1971. Purification and partim characterization of four proteins from human parolid ~aliva. Biochem. J. 123. 455-464. Fernandez-Sorensen A. and Carlson D. M, 1974. Isolation of a "prolinc-rich" protein from rat parotid glands following isoprotenerol treatment. Biochem. l~iophys. Res. Commun. 60, 249-256. Jacobsen N. 1970, In t,itro incorporation of [l*C]L-leucine into monkey (Macaca irus) parotid and submandibular gland proteins. Archs oral Biol. I5, 1163-1169. Jacobsen N, and S/Snju T. 1971. Molecular weight and amino acid composition of Macaca irus parotid alphaamylase. Scand. J. dent. Res. 79, 193-201. Levine M. J.. Ellison S. A. and Bahi P. O. 1973, The isolation from human parotid saliva and partial characterization of the protein core of a major parotid glycoprotein. Archs oral Biol. 18, 827-837. Meldolesi J. 1974. Dynamics of cytoplasmic membranes in guinea pig pancreatic acinar cells. I. Synthesis and turnover of membrane proteins. J. Cell Biol. 61, 1:-13. Meldolesi J., Jamieson J. D. and Paladc G. E. 1971. Composition of cellular membranes in the pancreas of the guinea pig. I. Isolation of membrane fractions. J. Cell Biol, 49, 109-129. Oppenheim F. G.. Hay D. I. and Franzblau C. 1971. Proline-rich proteins from human parotid saliva. I. Isolation and partial characterization, Biochemistry, N. Y. 10, 4233--4238. Robinovitch M. R.. Keller P. J., lversen L and Kauffman D. L. 1975. Demonstration of a class of proteins loosely associated with secretory granule membranes. Biochem. biophys. Acta 382, 260-264b. Wallach D., Kirshner N. and Schramm M. 1975. Non-parallel transport of membrane proteins and content proreins during assembly of the secretory granule in rat parotid gland. Biochim. biophys. Acta 375. 87-105. Williams B.L. and Keller P. J. 1973. Amylase and other protein components of Parotid saliva of the baboon, Papio anubis. Comp. Biochem, Physiol. 44A. 393-400. Zanetta J. P. and Vineendon G. 1973. Gas-liquid chromatography of the N(0)-heptafluorobutyrates of the isoamyl esters of aminoacids. 1. Separation and quantitative determination of the constituent amino acids of proteins. J, Chromat. 76, 91-99,

Fig, 2. Zymogen granules b.~fore (left) and after (right) extraction by dialysis ira alkaline hy~ohie buffer (electron micrographs × 50,000~.