biosynthesis of marsupial milk oligosaccharides: characterization and developmental changes of two...

Biochimica el BiophyMca Acta, 107/(1991) 79-85 © 1991 Elsevier .~ence Publishers B.V. 0167-4838/91/$03.50 ADON1S 0]67483891001283

BBAPRO 33859

Biosynthesis of marsupial milk oligosaccharides: characterization and developmental changes of two galactosyltransferases

in lactating mammary glands of the tammar wallaby, Macrop~ eugenii

M i c h a e l M e s s e r ! a n d K e v i n R . N i c h o l a s 2

t Depar~mem of Biochemistry, University of Sydney, N.S.W. and 2 Division of Wildlife and Ecology, CSIRO. Lyneham, A.C.7". (Australia)

(Received 16 July 1990) (Revised maauscript received 29 October !990)

79

Key words: Galactosyltransferase; Ofigosaccharide; Lactation; Tammar

Tananar wallaby ( Maeropus eugenii) mammary glands contort two galaetosyltranslerases of which the In, st, 4flGalT, is a UDP-galactose:N-acetylglueasaminyl/il ~ 4-galaetosyltransferase equivalem to the A protein of the lactose mjnthase of etaherima mmnmsd~ The second enzyme~ 3flGalT, is a UDP-galactose:lactose f l l ---, 3-galactosyltra~feras~ not previously identified in ~ gland~ of any species, which catalyses the formation of Gal,81 --* 3Galfli ~ 4Cde from hetose. The two enzyme activities, as well as the lactose synthase activity, have been c h a r a e t ~ with respect to the effects of pH, apparent K.~ values, effects of bovine and tammar a-lactallmmins, heat sensitivity and identity of products. Studies on the substrate specificity and heat sensitivity of the 3flGalT activity suggest that this enzyme may catalyse the fl-galactosylation of C, alfl l - , 3C, a l~ l - , 4GIc as well as of lactose. The activity of the 3flGalT, unlike that of the 4/~GalT, dmtges dramatically during the course of lactation in parallel with similar changes in the carbohydrate content of ~ m u a r milk.

lntreduefion

The milk of marsupials is unusual in that free lactose, the characteristic milk sugar of eutherian mammals, is generally only a minor component [1,2]. Instead, marsupial milk contains a variety of higl~er oligosaecba- rides [3,4] which, in the case of the tammar wallaby, Macropus eugenii, chiefly comprise a homologous series of f l l - - , 3 linked galactosides based on Galf l l - - , 3Galfll ---, 4Glc (3"-galactosyllactose) [5,6] and various derivatives thereof [7,8]. These oligosaccharides first appear in tammar milk on about day 4 post partum, after w~ch their concentration gradually increases [9], reaching a maximum of about 13% (w/v) by day 180. They then abruptly decrease in concentration and dis-

Abbreviations: 4~GalT, UDP-GaI:GIcNA¢ ~'l~4-galactusyltransferase; 3flGalT, UDP-Gal: Lac ~1 ~ 3-galactusyltransferase; tammar, tammar wallaby; TLC, thin-layer chromatography.

~2orrespondence: M. Messer. Department of Biochemistry, University of Sydney, Sydney, N.S.W. 2006. Australia.

appear by day 280, remaining absent from the milk until the end of lactation on about day 330 [10].

Very little is known concerning the mechanism by which these ofisosaccharides are synthesised within the marsupial mammary gland. In eutherian mammals (e.g., cow, human) the formation of lactose is catalysed by lactose synthase (EC 2.4.1.22), an enzyme which con- sists af two proteins, a fll --, 4-galactosyltransferase (EC 2.4.1.90,; A protein) and a-lactalbumin (B protein) [11]. In the absence of a- lacta lbumin the galactosyltransferase will utilise N-acetylglucosamine as aecep- mr, forming N-acetyllactosamine, but has very low affinity for glucose. The effect of a-lactalhumin is dramatically to increase the affinity of the galactosyltransferase for glucose, thus promoting the synthesis of lactose, and to inhibit its action on N-acetylglucosamine, a-Lactalbumins have been sho~m to be present in the milks of several species of marsupials [12-14] and it is likely that these a-lactalbumins act in the same way as eutherian a-lactalbumins; however, no marsupial galactosyltransferase has as yet been characterized in any detail. A galactosyltransferase which catalyses the formation of Galfll--* 3Galfll--* 4Glc from UDP-

80

galactose and lactose was recently identified in the albumen gland of a snail, and it was suggested that an enzyme with a similar specificity may occur in mammary glands of marsupials [15].

In this paper we report on the characteristics of two galactosyltransferase activities found in lactating mammary glands of a macropodid marsupial, the tammar wallaby, and on changes in these activities during the course of lactation.

Ma:erials and Methods

Materials UDP-D-[6-3H]galactose (16.0 Ci /mmol) was ob-

tained from Amersham Australia (Sydney). UDP- galaetose (dipotassium salt) and Tris-HCl were from Boehringer-Mannheim (N.S.W.). N-Acetyl-D-galacto- samine, N-acetyl-D-glucosamine, N-acetyllactosamine (synthetic), adenosine 5"-triphosphate (disodium salt), D-galactonic acid ¥-lactone, bovine a-lactalbumin (Type I), lactulose, mefibiose, methyl a-D-galactopyranoside, methyl fl-D-galaetopyranoside, methyl 3-0-fl-D-galacto- pyranosyl-fl-D-galactopyranoside, p-nitrophenyl /]-D- lactopyranoside and staehyose were from Sigma (St. Louis, MO). Lacto-N-tetraose, p-nitrophenyl fl-D- galactoside, p-nitrophenyl a-D-N-acetylgalactosamine and Galfll --, 6GIcNAc were generous gifts of Dr. Dirk Van den Eijnden (Vrije Universiteit, Amsterdam). 4 ' - Galactosyllactose was kindly donated by Dr. P.A.J. Gorin (Saskatoon). Galfll ~ 3Galfll ~ 4Glc [5], Galfll

3Galfll --~ 3Galfll ~ 4GIc [6] and a-laetalbumin [16] were isolated from tammar wallaby milk as previously described.

Animals Mammary glands were obtained from lactating tam-

mar wallabies (Macropas eugenii) at various stages of lactation and stored at - 8 0 ° C until required. The animals were maintained in large enclosures, about 250 m z, at the CSIRO, Division of Wildlife and Eeo!ogy, Canberra. Poach young were removed to reactivate the quiescent blastocyst [17] and parturition was assumed to occur 27 days later (day 0 of lactation). To age animals less than 7 days post partum accurately, their exact date of birth was determined by daily inspection of the pouch.

Preparation of enzyme extracts Samples of mammary tissue (about 100 mg each)

were reiaove0 fro_m_, the -g!tald and homogenised for 1 rain in 1 ral ice-cold 50 mM sodium cacodylate (pH 7.0) containing 0.5% Triton X-100, using an Ultra-Turrax Homogeniser. Each homogenate was centrifuged at 1000 × g for 10 rain, the pellet was discarded and the supernatant diaL, ted up to 8-fold with 50 mM sodium caeodylate (pH 7.0) containing 0.5~ Triton X-100. This

tissue extract was then immediately assayed for galactosyltrans ferase activity.

Galactosyltransferase assays The standard incubation mixture contained, in a

total of 100/tl, 20/zl of enzyme extract; 50 mM sodium cacodylate (pH 7.0)" 25 mM MnCl2; 2.0 mM ATP; 1.5 mM UDP-[3H]-galactuse (3.3 Ci/mol) ; and either 20 mM N-acetylglucosamine (for 4flGalT) or 200 mM lactose (for 3/~GalT). In addition, parallel incubations without acceptor were done to correct for hydrolysis of UDP-galactose. For the measurement of lactose synthase activity the standard mixture was modified by substituting glucose (20 mM) for N-acetylglucosamine or lactose and by the addition of 50 igg of bovine a-laetalbumin (500 /tg ml-~). The mixtures were incubated at 37°C for 20 rain and the reaction stopped by adding 50/ t l of 100 mM EDTA. The incorporation of [3H]galactose into oligosaccharide was determined by liquid scintillation counting following removal of resid- ual UDP-[~H]-galactose by filtration though anion exchange resin [18]. Protein was detem;,:.ned by the Lowry method [19] using bovine serum albu,'nin as the stall- dard. Enzyme activities are expressed as nmol product formed per rain per mg protein.

ATP was added to the assay medium to inhibit the activity of an enzyme, presumably nucleotide pyrophosphatase [13,20] which catalysed the hydrolysis of UDP- galactose. ATP at the concentration used (2 mM) inhibited the hydrolase activity by about 90~; higher concentrations were inhibitory to the galaetosyltransferase activities. ATP was a more effective inhibitor of the hydrolase than were N A D H or CDP-choline. The presence of ATP was essential for the assay of tissue obtained late in lactation, since the hydrolase activity increased markedly, from less than 2.0 nmol [IH]-galactose formed per rain per mg protein, prior to 255 days post partum, to over 15 nmol rain - l mg - I from 255 to 330 days post partum.

Heat inactivation Samples of enzyme extract were immersed in a ther-

mostatically controlled water bath at a given tempera- ture for various times fron: 0 to 60 rain and then immediately cooled to 4 ° C on ice. They were then assayed for galactosyltransferase or lactose synthas¢ activities as described above, except that for experiments with Galfll -o 3Galfll --, 4GIe the final aeceptor concentration was 10 raM.

Identification of products Thin-layer chromatography. An undiluted mammary

tissue extract was incubated at 37°C for 20 h with the standard incubation mix, after which 1 ~1 of the solu- tion was subjected to TLC on silica gel, using propan-2-

81

ol : acetone : 0.1 M lactic acid 4 : 4 : 2 (v/v) as solvent [21].

~H-NMR. For identification for the presumptive Gal/]l --+ 3Gal/]l ~ 4Glc, the standard incubation mix was modified by scaling it up 20-fold and by the use of unlabelled UDP-galactose and the addition of 40 mM D-galactonic acid ~/-lactone, an inhibitor of rat mammary/~-galactosidase [22]. After 20 h at 37°C the incubation mix was f'dtered through a colunm (0.5 × 8.0 cm) of anion exchange resin (see above). The ehiate was freeze-dried, dissolved in 0.5 ml of water and filtered through a column of Sephadex G-25, Supertrme, 0.7 × 140 cm which had previously been calibrated with a mixture containing galactose, lactose, Gal/~l --, 3Gal/~l --* 4GIc and Gal/~l --+ 3Gal,81 -+ 3Gal/] --+ 4GIc (1 mg of each). Carbohydrates were ehited at 1 ml h - I with water and monitored by the phenol-sulphuric acid method [10]. Fractions containing the presumptive Gal~81 --, 3Gal~l --, 4Glc were examined by TLC and those which were not contaminated with lactose were pooled and freeze-dried. The product, (1.1 mg) was identified by IH-NMR using a Varian XL-400 wide bore NMR spectrometer; spectra were recorded in D20 at 298 K.

30

E 7 E o

g _1 3

. / ' \

o

10

I ! I 05L. 6 7

pH Fig. 1. Effect:~ of pH or, galactosyhrunsferase and lactose syathase activities of extract of tammar wallaby r-ammary glands obtained at 150 days po~t panum. 0, o, 3~SGalT (acceptor, lactose), It, 0, 40GAIT (acceptor, GIcNAc): A. e,. lactose synthase (acceptor, glucose). Closed symbols, sodium cacodylate buffers. Open symbols, N-2*hydrory-

ethylpipesazine-N '-2-ethanesulfonate (Hepes) buffers.

R e s u l t s

Characterization of 4[J- and 3[3-GalT activities; lactose synthase

Preliminary experiments with mammary glands obtained at about 150 days post partum (mid-lactation) showed that extracts of the glands exhibited at least two galactosyltransferase activities, 4~GalT and 3flGalT, which catalysed the transfer of labelled galactose from UDP-galactose to GIcNAc and to lactose, respective!y. The 4,SGalT activity was optimal at about pH 7.2, whereas 3/3GaiT activity had a pH optimum of 6.5 (Fig. 1). Both activities had an absolute dependence on Mn 2+ and were 97~ inhibited by 30 mM EDTA (data not shown). The 4~GalT activity was optimal at 20 mM GIcHAc with substrate inhibition evident at higher con- centratious, whereas optimal 3~GalT activity required relatively high concentrations of lactose (Fig. 2). The respective values for the apparent /t'm'S of 4/]GaiT and 3/]GAIT calculated from Lineweaver-Burk plots (data not shown) were 8.1 4-0.44 mM (mean 4-S.D., n = 4 )

for GIcNAc and 46 4- 3.8 mM (mean 4- S . D . , n = 3) for lactose.

When glucose was used as acceptor instead of GlcNAc or lactose, the galactosyltransferase (i.e., lactose synthase) activity was almost undetectable except either at very high glucose concentrations or in the presence of a-iact~,lbumin (Fig. 2). In the absence of a-lactalbumin the apparent K m for glucose was 644 4- 55 mM (mean 4-S.D., n = , ) ; bovine a-lactalbumin (500 pg ml - I ) marke~41y stimulated the lactose synthase activity in-

sofar as it lowered the apparent K m for glucose to 5.1 + 0.59 mM (mean + S.D., n = 3). The optimum glucose concentra0vn in the presence of bovine a-

• 0 §0 100 150 2 0 o

A c c e p t o r ( m M ) Fig. 2. Galactosyltransferase and lactose synthase activities as a function of acceptnr concentration. ! NoAcetylglnoesamin©; Q, laclose; A, glucose in the prese.~ce of a-lactalbumin; ~, glucose alone. Values for VmM were calculated from Linewcuver-Bnrk plots of data obtained at substrate concentrations at which there was no substrate

inhibition (data not shown). For other details see Methods.

82

lactalbumin was about 20 raM, substrate inhibition being evident at higher concentrations (Fig. 2). Com- parison of bovine and tammar a-lactalbumins showed that the former was significantly more effective than the latter in stimulating lactose synthase activity; the apparent Kra values were 120+ 18 pg nd -z for bovine and 340 + 50 pg ml - ' for laminar a-lactalbumin (mean + S.D., n=3).

The 4,SGalT activity was strongly inhibited by bovine a-lactalbumin, 50% inhibition being observed with 20 pg m l - l (Fig. 3). Tammar a-lactalbumin was less effec- five, 50% inhibition requiring 380 pg m1-1. Neither bovine nor tammar a-lactalbumin had any effect on the 3flGalT activity (Fig. 3).

Heat inactivation studies, in which the tissue extract was pre-heated at various temperatures prior to assay of enzyme activities, showed that the 3flGalT activity was considerably less stable than the 4,SGalT activity. Heat- ing for 30 rain at 390C caused complete loss of 3~GalT activity, but had no effect on 4]]GalT. A loss of 50% of the original activity was observed after 20 rain ;)eating at 36°C for 3fiGalT and after 32 rain heating at 4 4 ° C for 4~GalT (Fig. 4). The presence of 25 mM MnCl 2 almost completely protected 3~GalT against heat inactivation at 36°C (11% loss of activity over 60 rain), but did not protect 4flGalT against inactivation at 44°C. The lactose synthase activity was inactivated at the same rate as the 4flGalT activity during heating at 4 4 ° C (Fig. 4).

Identification of products When the incubation mix was examined by TLC

following prolonged incubation under standard assay condition.~ and using GlcNAc as acceptor, the only detectable product co-chromatographed with N-acetyllactosamine. The major product with lactos~ as acceptor (3~GalT activity) co-chromatographed with Galfll

Io0 : : : : : : : : : : : : : : : : : : : : : : : : : : : :

o \ o

:--~ 4I r:

~ , o \

o 2;0 s;o ;,~o 1ooo

a-Lactalburnin (pg rnl - t )

Fig. 3. Effec~ of bo-vine 01) and tarnmar wallaby ([:3) a-]actr.Jbumins on activities of 4~GalT (substrate, GIcNAc; solid lines) and 3flGalT

(substratc, lactose; broken fines).

1 0 o

8 0

60

~ a0

~ 2o

o 1

o 20 0 60

Time (rain)

Fig. 4. Heat inact/va!ion o f galactosyltransferaso and lactose synthaso a~::-vitie~ at ~,4°C or 3 6 ° C . ~ , N-Aoetylglucosamine (4flGalT); A, glucose (with a-lactalbumin, lactose synthas¢); @, lactose (3flG~IT); o , Ga l f i l ~ 3GaIf l l ~ 4GIc (presumptive 3flGalT). Note logar/thm~c

scale on ordinate.. For details, see Methods.

3Galfll ~ 4Glc and no higher homologues oi this m - saccharide, such as di- or tri-galactosyUacto~, could be detected; however, small amounts of free glucose and galactose were formed, presumably through the action of/~-galactosidase on lactose. When glucose was used as acceptor, in the presence of bovine a-lacUalt umin, the only product co-chromatographed with lactose.

The trisaccharide product obtained when lactose was used as acceptor was isolated from a scaled-up incubation mix (see Methods) and investigated by 400-MHz 'H-NMR. Its spectrum was essentially identical with that of authentic Ga i t1 --* 3GalOl --* 4Glc izolated from tammar milk whose structure had b,~n estabfished by 13C-NMR and other methods [5]. In both spectra, signals diagnostic of a G a l f l l - , 3 linkage [15,23] were observed at 4.62 and 3.92 ppm, in addition to signals at 4.67, 4.~1 and 4.20 ppm which are characteristic of Galfll ---, 3Galfll -o 4Glc [15,231.

Substrate specificity of 3flGalT activity A number of galactosides were compared with lactose

as acceptor substrutes, all at 5 mM (Table I). Lactose was the best substrate, followed by its p-nitrophenyl derivative. Three other substrates, viz. Galfll --, 3Galfll -~ 4Glc, lacto-N-tetraose and Gal/~l - , 3Oalfll --* 0-Me, all of which have fi-Gal in a 1 ~ 3 linkage, supported activities ranging from 22-38% and seemed to be better substrates ".hen those with fl-Gal in a 1 ~ 4 linkage, such as Gal~81--* 4Galfll--* 4Glc or N-acetyllactosamine. Surprisingly, methyl a-galactoside was a better

substrate than methyl /~-galactosidc. Gaiactose and GalNAc were poor substrates. The observation that Galfll ~ 3Gal~81 --* 4Glc was a substrate w ~ of consid- erable interest, since this trisaecharide was the oligo- sacchar/de product when ~actose was used as accepter. To obtain information on whether more ~haa one ,81 3-galactosyltransferas~ might be involved in the galactosylation of these two substrates, the rates of inactivation of the act;.vities towards lactose and Gal/il ---,3Galfll--,4Glc during pte-hcating at 3~oC were compared; it was found that these rates were essentially identical (Fi~ 4).

Developmental changes Mammary glands from a total of 34 animals which

had been lactating tot various periods trom 1 to 330 days were assayed for galactosyhransferase activities (Fig. 5). 4~GalT was present at parturitiou, its specific activity averaging 3.7 + 0.32 nmol rain - t mg - t protein (mean + S.D., n = 3) during the first 4 days Fost partum. By contrast, 3flGaiT was virtually undetectable at this stage, its specific activity being less than 0.20 nmol rain - t rag - t during the first 4 days. A significant in- creaze in 3flGalT activity was first detected on day 6 (0.60 mnol rain ~t mg - i ) and both GaIT activities then gradually increased over the next 90 days. From day 90 to day 225 the activities of both enzymes, although variable, remained elevated at 13 =i: 4.5 and 20 __. 6.8 mnol rain -~ mg -~ (mean _+ S.D., n = I3) for 4/~GalT and 3~SGalT, respectively. The 3~SGalT activity then fell very sharply, becoming undetectable by 270 days post partum, unfike the 4/~GalT which fell only moderately to 8.1+3.0 nunol rain -~ mg - t (mcan+S.D., n = 6 ) during the final period from 270-330 days. To de-

T A B L E I

Acceptar specificity of tammar wallaby nmmnmry gland galactc.syltransfero.~e

~81 ~ 3-

Accepter (5 mM) Relative activi,'y

Gal,81 ~ 4Glc (lactose) 100 Gal#l ~ 4 G ~ .4 0-pN,h 52 Galfll --* 3G'~1~81 --, 4GIc (3 "-galactosyllactose) 38 Galfl1.4 3GlcNA~1.4 3Galfll ~ 4GIoOacto-N-tetraose) 28 G ~ m . ~ 3Gal,q:! ~ .O-Me 22 Gal/~l ~ 4Fro (lactulose) 18 Galal ~ O-Ma 15 Gal.BI ~ 4Gal~Bl ~ 4GIc (4'-galactosyilactose) 6.8 GaltS1.4 4GIcNAc (N-acety]lacto~;amine) 6.2 Galt81 ~ 0-Ma 5.1 Gala] ~ 6Galal ~ 6GIc~ .4 2Fru (stachyose) 4.0 Gal 3.2 Oal/~1-4 6GIcNAc 3.1 GalNAca1.40pNph 2.5 GalNAc 1.5 Gala] ~ 6GIc (melibiose) < l Galfll --* 0-pNph <1

E 7 c E o

>,

t -

O

83

(a)

t 0 •

e (b)

30

2Oie e • • o

lC

0 100 200 3co

Time past par tum (days )

• | I n awl

• I 1 ~11 • ~ a e

e~(c ) • "

• 4 ~ • • |

2~" • • ee• e ~ a ~ B .

O 5 10 15

8 e'• • • I

~ig. 5. Ga lac tosyhra~fe rase activitief at v a n • u s t imes post partum. (~.) 4/]GAIT activity (b) 3BGaIT activity and (c) inset showing changes in 4/~GalT (11) and 3/3GAIT (e) activities during the In'st 14 days

post partum.

termlne whether the absenc~ of 3flGalT activity late in lactation might be due to an inhib;.tor, tissue extract from a mammary gland obtained at 330 days post partum was added to a 9~day tissue extract; no inhibition of the 3~GaiT ac'civity of the 90-day extract was observed.

Galactosyltransferase activities in mammary tissue from a eutherian mammal

4,SGalT and 3jSGalT activities were measured in an extract from the mammary gland of a lactating labora- tory mouse killed at 12 da~/s post partum. The 4jSGalT activity was 7.6 nmol ra in- ' mg - t protein, but no 3flGalT activity could be detected.

Discussion

Our results show that tammar wallaby mammary glatlds contain at least two galactosyltransferases. The first enzyme, 4pGa lT , resembles the U D P - galactose : N-acetylglucosaminyl /31 --, 4-galactosyltransferase of eutheriar, mammary glands, notably in its inhibition by a-lactaibumin [11], and our results are consistent with the view that the lactose synthase activity observed in the presence of a-lactalbumin was due to this galactosyltransferase Given that tammar wallaby milk~ like eutherian milk, contains a-lactalbumin [13], the synthesis of lactose in the tammar mammary gland appears to proceed by the same mechanism as that found in the eutherian mammary gland [1i,24].

Tammar a-lactalbumin was ]~ss effective than bovine a-tactalbumin, both as an inhibitor of 4flGalT activity

84

and in promoting lactose synthesis. Although somewhat surprising, this obsereation is consistent ,.vith pre~,io~ studies suggesting that a-lactalbumins and galactosyltransferases do not exhibit co-adaptation to each other within a species [25,26]. It may be noted that the observed K m values for bovine and tammar a- lactalbumins (120 and 340/~g ml - l , respectively) are of the same order as previously published K m values of bovine and porcine galactosyltransferases for the a- lactalbumins of various species; these range from 60 to 373/tg ml -~ [26].

The second enzyme, 3/~GalT, is a UDP-galactose: lactose/~1 - , 3-galactosyltransferase which catalyses the formation of Galp l - , 3Gal//1 --, 3Glc from UDP- galactose and lactose. //-1 --, 3-Galactosyitransferases which use lactose as acceptor have been recently detected in human kidney microsomes [27] and in extracts of the albumen gland of the snail, Lynmaea stagnalis [15]. However, the activities reported in the present work (up to 32 nmol rain-~ rag-~ protein) are higher, by several orders of magnitude, than those reported for either the human or the snail enzyme. Of various galactosides tested, lactose was the best acceptor for galactose; in this respect 3~GalT resembles the snail enzyme but differs from the human enzyme for which methyl p-galaetoside and several other//-galactosides are better snbstrates than lactose [28]. It is not as yet possible to decide whether 3/~GalT is identical with the snail enzyme as neither of the two enzymes has been purified.

The presence of these two galactosyltrausferases, 4/~GalT and 3/~GalT in the tammar mammary gland indicates that, in vivo, Ga l / /1 - ,3Gal /~ l - - ,4Glc is formed from UDP-galaetose and glucose by the sequential actions of lactose synthase and 3/~GaiT. The question ar is~ as to whether the higher homologues of this trisaccharide, some of which are found in the milk at even greater concentrations [10], are synthesised by 3pGalT or by one or more additional /~1-* 3- galactosyltransferases. Our heat inactivation experiments suggest that the conversion of Gal/31 --, 3Gal//1 --, 4Glc to the presu~aptive Gal/31 --* 3Gal~01 --, 3Gal/~l --* 4Glc (digalaetosyilactose) is eatalysed by 3/~GalT and not by a separate enzyme. In that case it would be surprising if the further conversion of di- to tri-, tetra- galactosyllactoses, etc., required a separate enzyme. TLC of the incubation mix failed to show products other than Ga l /H-* 3Galpl - - ,4Glc , but it is likely that lactose, at the high concentrations used under the standard assay conditions, would prevent the formation of any products beyond Gall//1 --* 3Galpl --~ 4Gie. Never- theless, the question of whether only one enzyme catalyses the sequential p-galactosylation of a series of galactosyllaetoses must remain open until the enzyme has been isolated in pure form. In addition it should be noted that tammar wallaby milk contains oligosaccha-

rides in which galactose is linked to .~'-aeetylglueosa- --mine via a ,8] --, 4 linkage [29] and it remains to be established whether this linkage is formed by 4pGalT or by a separate/~1 - , 4-galactosyltransferase.

The 3,SGalT activity, unlike 4pGalT, did not appear in the tammar mammary gland until several days after parturition and disappeared well before the end of lactation (Fig. 5). This developmental pattern is similar to, and presumably correlated with, the pattern of ap- pearance and disappearance of higher oligosaccharides from the milk and with accompanying quantitative changes in the milk carbohydrate content. Higher oligc~ saccharides are first observed in the milk on day 4 post parzum [9], i.e., very close to the first detection of 3~GalT activity in the gland on day 6; similarly, the disappearance of these oligosaccharides from the milk [10] and the loss of 3,8GalT activity were both complete by day 270 post partum. The concentration of carbohydrate in the milk, which reaches a peak between 170 and 200 days post partum [9], also appears to be related to the elevated levels of 3/~GalT (cf. Fig. 5).

Most of the changes, both qualitative and quantitative, in the carbohydrates of tammar wallaby milk are thus explicable in terms of changes in 3~OaiT activity. A separate mechanism must, however, supervene towards the end of lactation when lactose, as well as the higher oligosaccharides, disappears from the milk [10]. This is not due to a cessation of a-lacta!'-umin synthesis since a-lactalbumin is secreted throughout lactation [13]. The present results exclude the further possibility that it is due to absence of 4BGalT activity, since this enzyme was present throughout lactation (Fig. 5). Our fmding of high UDP-galaetose hydrolase activity in mammary tissue obtained late in lactation supports the suggestion [13] that lactose formation stops at this stage because of extensive hydrolysis of the substrate, UDP-galactose, by nucleotide pyrophosphatase. The functional significance of this phenomenon is not immediately apparent.

In summary, our results demonstrate that tammar wallaby m a m m a r y g lands conta in a /~1--+ 4- galactosyltransferase as in eutherian mammals as well as a ffl ~ 3-galactosyltransferase whose activity appears to determine the concentration of some, if not most of the higher oligosaccharides of tammar wallby milk. It will be of great interest to discover how the biosynthesis of the latter enzyme is controlled in rela- tion to the control of other products of the tammar mammary gland, such as 4pGalT, late lactation protein, a-laetalbumin and caseins [30-32].

Acknowledgements

This work was supported by the Australian Research Council and by the CSIRO/Univers i ty of Sydney Col- laborative Research Fund. The work was begun at the CSIRO Division of Wildlife and Ecology during the

85

t enu re o f a Specia l S tudies P r o g r a m m e for wh ich leave h a d k ind ly been g r a n t e d to M . M . b y t he U n i v e r s i t y o f Sydney . W e t h a n k Dr . W . A . B u b b for p e r f o r m i n g the N M R spec t roscopy a n d Drs . L .A. H i n d s a n d C . H . Tynda l e -B i scoe for ass i s tance wi th an ima l s .

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biosynthesis of marsupial milk oligosaccharides: characterization and developmental changes of two...

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