GlycobWogy vol. 2 DO. 4 pp. 337-343, 1992
Developmental sialic acid modifications in rat organs
Elaine A.Muchmore Hematology-Oncology Division, Department of Medicine, University of California at San Diego, VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, USA
Key words: A/-acetylneuraminic acid/A'-glycolylneuraminic acid/ sialic acids
Introduction Sialic acids, which are often the terminal carbohydrate moieties on cell surface glycoproteins and glycolipids, are in a unique position to have effects on cell-cell recognition. Sialic acids can be modified by the addition of various groups at the 4, 7, 8 and 9 positions. A family of naturally occurring compounds has been described which now has > 30 members (Schauer, 1982; Varki, 1992). One modified sialic acid is A/-glycolylneuraminic acid (Neu5Gc). This sialic acid is formed by conversion from the parent molecule, JV-acetylneuraminic acid (Neu5Ac), by CMP-yV-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (Shaw and Schauer, 1988; Muchmore etal., 1989). We have previously reported (Muchmore et al., 1987) that the expression of Neu5Gc in Sprague-Dawley rat colonic mucosa is developmentally regulated. It is present prenatally, disappears rapidly in the postnatal period, and then reappears. The glycoconjugates involved were not determined in this study. Several investigators have reported developmental changes on gangliosides from selected rat organs. G ^ has been determined to be the major ganglioside in rat small intestinal cells (Bouhours and Bouhours, 1983) and the sialic acid on G ^ changes from Neu5Ac at birth to Neu5Gc at the time of weaning. Furthermore, a shift from sialylation to fucosylation of rat small intestinal glycoconjugates by the weaning phase has been demonstrated (Taatjes and Roth, 1988, 1990, 1991). Developmental differences in the ganglioside composition of rat stomach mucosa have also been described (Bouhours et al., 1987). In this organ Oxford University Press
Results Neu5Gc expression is developmentally regulated in all tissues examined We have previously shown that Neu5Gc expression in rat colonic mucosal cells is developmentally regulated. In this study, sialic acids from the ganglioside fraction of multiple organs of littermates were assayed. The results in Table I are the sialic acids fractionated from the gangliosides of various organs from two littermates at each time period, expressed as nmol/mg protein in the membrane pellet. Sialic acids in the cytosol and glycoprotein pellet contribute < 10% of the total sialic acids, and are below the detectable range for many time points (data not shown). As can be seen in Figure 1, which graphically depicts sialic acids extracted from thymus gangliosides, there is a striking perinatal increase of total sialic acid/mg protein extracted from the ganglioside fraction. A similar pattern occurs in all organs sampled between days 1 and 3. No similar increase in the expression of either sialic acid was seen, although the litters were followed to day 28 (complete data not included). Although the absolute amount of Neu5Ac is greater at all time points, the increase in Neu5Gc is parallel. The majority of the sialic acid is on gangliosides Because sialic acids could not be quantitated in fractions other than the gangliosides in the time course study, a more detailed analysis of organs from 10 animals of postnatal days 1 and 6 was performed. Organs were fractionated into low molecular weight cytosolic compounds, soluble proteins, gangliosides and membrane proteins. As can be seen in Table II, >99% of the sialic acid on day 1 is present in the ganglioside fraction. Even with the larger number of animals, the amount of sialic acid in the membrane protein fraction is small in all organs. The amount of sialic acid in the ganglioside fraction decreases substantially by 337
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The changes in expression of sialic acids in Sprague-Dawley rats in the prenatal and early postnatal time period have been examined in multiple organs, both visceral and nonvisceral. In all organs examined, there is a dramatic increase in both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) shortly after birth. The bulk of the sialic acid is present in the ganglioside fraction in all tissues examined. As total amounts of sialic acid present in gangliosides decrease, the proportion present in the low molecular weight cytosolic fraction increases. A curious observation is that Neu5Ac hydroxylase activity is present at the time of the increase in sialic acid, but its activity does not correlate with Neu5Gc expression after the early postnatal period. This implies that Neu5Gc expression has another level of regulation besides CMP-Neu5Ac hydroxylase activity.
GM3 and GQ3 are the major gangliosides at birth, but GMI, G ^ , fucosyl GM1 and a blood group B-active ganglioside are added during postnatal development. In this study, the oligosaccharide portion of membrane and cytosolic structures in multiple rat organs (spleen, thymus, liver, stomach, small intestine and colon) have been examined at multiple early postnatal days, using recently described techniques for the analytical quantitation of sialic acid modifications (Hara et al., 1989). Sialic acids were localized to membrane fractions, soluble glycoproteins and low molecular weight cytosolic elements on selected postnatal days. Assays of CMP-Neu5Ac hydroxylase were carried out at all time points. The intention of the study was to determine the major oligosaccharide structures which contain the sialic acids and whether the previously seen developmental changes extended to non-visceral organs. A related question was whether Neu5Gc expression correlated with CMP-Neu5Ac hydroxylase activity.
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Table I. Sialic acids released from gangliosides with sialidase and acetic acid, and quantified using derivatization with DMB over a TSK-ODS-120T column. The upper number for each day is Neu5Ac, the lower is New5Gc, expressed as nmot/mg protein in the organ pellet Small intestine
Day
Spleen
-1
1771 61
499 75
24 4
1
1093 93
1990 323
2
837 180 52 8
4
1.4 0.2
9
5.0 1.3
11
8.4 2.4
21
6.6 2.7
Colon
Liver
546 61
149 99
173 103
741 58
5450 263
105 14
429 22
3897 860
66 6
3244 457
416 109
23 2
43 9
128 10
406 48
507 71
156 10
n.d.
n.d
.
26 0.4
32 4.9
5.3 1.8
44 6.9
19 8.6
5.6 0.03
0.6 0.4
10 1.2
14 4.8
10 0.7
2.9 0.3
2.0 1.2
11 4.4
2.9 1.3
2.7 0.4
0.8 0.1
4.0 1.8 58 24
n.d., not determined. DMB, l,2-diamino-4,5-methylenedioxybenzene.
day 6. In this analysis, Neu5Gc is the predominant sialic acid in all organs except small intestine on day 1, implying that there is variation of Neu5Gc expression between animals. The percentage of total sialic acids in the cytosol increases on day 6 compared to day 1 As shown in Table n, the decrease in total sialic acid in the ganglioside fraction on day 6 is paralleled by an increase in total sialic acids present in the cytosol at this time. The relative increase in total sialic acids varies in each organ. The organ with the smallest change (8-fold increase) in the percentage of total sialic acids present in cytosol is the spleen, but other organs (such as the liver and colon) have a 130- to 175-fold increase between days 1 and 6 (Figure 2). The consistent trend in each organ, except the spleen, is that the predominant increase in sialic acids occurs in the ethanol supernatant. The ratio of Neu5Ac and Neu5Gc in the cytosol also varies with the organ. In all organs tested, the amount of Neu5Ac in the cytosol, in both the ethanol supernatant and pellet, is greater on day 6 compared to day 1. CMP-Neu5Ac hydroxylase activity does not correlate with Neu5Gc expression CMP-Neu5Ac hydroxylase activity is present in all organs tested in the prenatal samples (gestational day 20). After postnatal day 1, enzyme activity is consistently present in spleen and thymus, but is not detectable in small intestine or colon until day 13, and in stomach until day 28 (Figure 3). Even at day 28, activity is not detected in the liver. The level of enzyme activity does not correlate closely with Neu5Gc expressed in the ganglioside fraction. The relatively high levels in the prenatal and early postnatal period correlate with the dramatic increase in Neu5Gc 338
The ganglioside pattern changes slightly between days 1 and 6 in most organs As seen in Figures 4 and 5, both the 'a' series ( G ^ , GM2, G M h GDia. Gria) and the 'b' series (GD3, GD2, G m b , GJU,, GQ,b) are expressed at both days 1 and 6. All organs at both times have a strong, usually double, band at GM3. The chromatographic profile changes slightly in most organs between days 1 and 6. Gangliosides extracted from spleen on both days are GD|a, GD3 and GM3. In thymus, both GD3 and GM3 are seen on day 1, but only Gj^ is seen on day 6. Small intestine has Gr| b , GD3 and GM3 on day 1, but only GM3 could be seen on day 6. In colon, G Tlb , GD3 and G ^ are seen on day 1 and, on day 6, GQ]b and G D | b are also present. In stomach, the G Tlb band seen on day 1 is not seen on day 6, but on both days GD3 and GM3 are seen. In the liver, GD,a, GM1 and GM3 are seen on day 1; GD,b, GT,b appear on day 6. From this pattern, it appears that glycosyltransferases are expressed at both time points and that developmental modifications of gangliosides continue to occur. Therefore, the decrease in the total amount of ganglioside, which occurs in all organs, is not due to a difference in either the major ganglioside expressed or to a change in the developmental modifications. The sialic acids of the membrane pellet glycoproteins do not increase as those on gangliosides decrease Sialic acids released from the residual pellet after extraction with chloroform and methanol are not increased at day 6, when there is a dramatic decrease in sialic acids on gangliosides. As seen in Table I, there is not a substantial increase in either Neu5Gc or Neu5Ac on day 6 in the glycoprotein fraction in any organ. Similarly, sialic acids on glycoprotein fractions from later gestational days, when CMP-Neu5Ac hydroxylase is detectable, are not increased (data not shown).
Discussion The diversity of the sialic acids has been well established. The appreciation of the complexity of the processing has encouraged investigation regarding its biological significance. Despite the clear developmental changes noted in various organs, their biological role remains unclear (Schauer, 1982; Varki, 1992). Previous studies regarding the developmental regulation of Neu5Gc have been carried out in isolated organs of various animals (Bouhours and Bouhours, 1983, 1988, 1989; Bouhours et al., 1987; Taatjes and Roth, 1988, 1990, 1991). Many careful studies have been done on rat small intestine mucosal cells. These have showed changes in the glycoconjugates which occur both in the early postnatal period and at the time of weaning. An obvious event which could be involved in intestinal changes at these time periods is exposure to environmental organisms and their by-products. It has been demonstrated that
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7
Stomach
Thymus
during this period. However, the highest level detected is in the thymus on day 9, which is associated with only a modest rise in Neu5Gc/mg protein. In addition, the activity levels seen between days 11 and 28 are comparable to those in the prenatal and early postnatal period, but there is a clear difference in Neu5Gc expression during this period. Therefore, it appears that factors other than CMP-Neu5Ac hydroxylase activity control Neu5Gc expression.
Rat developmental sialic acid modifications
£
4000
O
o Q. 3000-
E o ea
Neu5Gc
2000 -
NeuSAc
(0 CO
1000-
perinatal
day
Fig. 1. Perinatal rat organ sialic acids. Ganglioside extracts were prepared (Materials and methods) from rat thymus from two animals at each pennatal day. Sialic acids were released with sialidase and acetic acid, and quantified using derivatization with DMB over a TSK-ODS-120T column. The open circle is Neu5Ac, the closed circle Neu5Gc, expressed as nmol/mg protein in the organ pellet. See Table II for quantification.
Table n . Sialic acids (nmol/mg protein) in cellular compartments from Sprague—Dawley rat organs on postnatal days (D) 1 and 6 Cytosol
Pellet
NGc
Ganglioside
Glycoprotein
Ethanol pellet
Ethanol soluble NAc
NGc
NAc
NGc
NAc
NGc
NAc
Spleen
Dl D6
0.5 0.6
1.6 1.2
0.09 0.7
0.05 0.99
0.05 0.3
0.22 0.7
1337 90
702 250
Thymus
Dl D6
0.5 1.2
0.04 2.9
0.44 0.13
0.02 1.4
0.08 0.4
0.16 0.6
1435 116
181 306
Small Intestine
Dl D6
0.15 1.0
0.01 18.9
0.21 0.5
0.06 2.9
0.01 0.3
0.03 0.6
28 164
90 351
Colon
Dl D6
2.0 1.5
0.16 31.2
1.0 2.5
0.19 3.1
0.2 0.02
1.1 0.1
1907 38
503 71
Stomach
Dl D6
0.63 1.09
1.62 11.4
0.15 0.14
0.06 4.8
0.33 0.06
0 27 0 26
704 79
259 174
Liver
Dl D6
0.02 0.4
0.02 0.60
0.01 0.2
0.01 0.9
0.002 0.002
0.01 0.04
24 39
29 9.9
NGc, A'-glycolylneuraminic acid. NAc, A'-acetylneuraminic acid.
the acid and non-acid glycosphingolipids of stomach, small and large intestine, and faeces of germ-free and conventional rats are chemically similar, with the exception that Neu5Gc-GM3 ' s found in the stomach of conventional, but not germ-free rats (Gustafsson et al., 1986). In the current study, sialic acid expression in various compartments of intestinal organs and spleen, thymus and liver from conventionally fed Sprague—Dawley rats has been studied. In Sprague-Dawley rats, there is a striking perinatal increase in sialic acid production in all organs tested on
days 1-2. More than 99% of the sialic acids during this period are in the ganglioside fraction. Gangliosides injected into plasma can be taken up by distant organs such as the liver (Ghidoni et al., 1987). Therefore* a concern would be that the environmental stimulus delivered to the intestinal organs stimulates ganglioside production in these organs with subsequent export of gangliosides to non-intestinal organs. Although this cannot be ruled out with certainty, the rapidity of the change in all organs tested and the observation that the gangliosides from each organ 339
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o E
E.A.Muchmore
(0
DAY 6 DAY1
30
percent Fig. 2. Percentage of total sialic acid in cytosol. Organs from 10 littermates were fractionated into low molecular weight cytosolic compounds, cytosolic proteins, membrane proteins and gangliosides (Materials and methods). The sialic acid in the cytosol is expressed as the percentage of total sialic acid. 1, spleen; 2, thymus; 3, small intestine; 4, colon; 5, stomach; 6, liver.
28 21 17 15 13
O
11
0 colon HI small bowel
CD
9
stomach
[I] thymus H spleen
7 4 2 1 -1 0.00
0.10
0.20
p m oIes/mi n/m g Fig. 3. CMP-Neu5Ac hydroxylase activity in cytosolic fractions. Cytosolic extracts from each organ were prepared and incubated with CMP-[9-3H]Neu5Ac (28.9 Ci/mmol) and co-factors as described in Materials and methods. The reactions were quenched in 90% ethanol and the sialic acids in the supernatant separated by descending paper chromatography in n-butand, n-propanol and 0.1 N HC1 (1:2:1). Radioactivity was monitored after cutting 1 cm strips. Activity is expressed in pmol/min/mg protein in the fraction.
have distinctive patterns make it unlikely. This implies that the increase in sialic acids is part of a developmental sequence that is independent of environmental factors. There is a perinatal increase in Neu5Gc which parallels the increase in Neu5Ac. In this study, the two assays of day 1 340
gangliosides demonstrated a variation in the predominant sialic acid in the gangliosides. There are several possible explanations for the differences in the proportion of Neu5Gc to total sialic acid in the early postnatal period. First, all membrane-bound sialic acids may not be accessible to release by either acid or sialidase.
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20
Rat developmental sialk add modifications
- G M3 ~ G M1 "GD3 ~GD1a
- GT1b
~
G
M3
-GD3 ~ GD1a "~GD2 -GD1b -Gjib ~ GQ1b
i Fig. 5. TLC of gangliosides extracted from rat organs on day 6. Gangliosides were prepared from membrane pellets as described (Materials and methods) and analysed on silica gel 60 pre-coated HPTLC plates (Merck) developed in chJoroform/methanol/water containing 0.2% CaCl2, 60:40:9, v/v. Samples are pooled from organs harvested from 10 animals. Lane 1, spleen; 2, thymus; 3, small intestine; 4, colon; 5, stomach; 6, liver.
Although this does not seem to be a problem with the current study because sialic acids were released with both sialidase and acid from all samples, it may have been an issue in an earlier paper (Muchmore et ai, 1987). Second is the less likely possibility that glycosyltransferases have variable expression. The feedback loops are not known, and although sialic acids do not appear to accumulate in the cytosol of perinatal specimens, this does not prove that there is no block in the formation of gangliosides. Free sialic acids in the cytosol may be degraded rapidly. Third, it is possible that the level of CMP-Neu5Ac hydroxylase activity varies between animals. Thus, if a few animals have high levels, there could be large amounts of Neu5Gc in the pool. This possibility seems the most likely because this variation would represent a subtle change in timing of the already observed decrease in CMP-Neu5Ac hydroxylase activity on day 1 in most organs. However, resolution of this issue requires analysis of individual animals, which must await more sensitive assays for the CMP-Neu5Ac hydroxylase and for the measurement of sialic acids. Between days 1 and 6, there is a change in distribution of sialic acid within the cell, such that a higher percentage is present in
both the low molecular weight cytosolic pool and in soluble proteins. The increase in sialic acid in the cytosol is primarily Neu5Ac. During this same interval the amount of sialic acid in the gangliosides decreases. The sialic acid in the cytosol may be explained by release from lysosomes of sialic acids from recycled gangliosides. It is likely that the cytosolic Neu5Gc is recycled in the intestinal organs. At day 6, there is no detectable CMP-Neu5Ac hydroxylase activity in these organs, and because this enzyme is the only known pathway for conversion of Neu5Ac to Neu5Gc, any Neu5Gc present in the cytosol would be recycled. • The lack of correlation between CMP-Neu5Ac hydroxylase activity and expression of Neu5Gc in the ganglioside fraction is unexpected. A correlation in AVN rats, which convert to GM3(Neu5Gc) in the weanling period, between CMP-Neu5Ac hydroxylase activity and CMP-Neu5Gc in the cytosol has been demonstrated (Bouhours and Bouhours, 1989), and a microsomally bound enzyme which hydroxylates intestinal glycoproteins in mutants without intestinal CMP-Neu5Ac hydroxylase activity. Of interest in Bouhours' study is the fact that AVN mutants without intestinal CMP-Neu5Ac hydroxylase activity 341
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Fig. 4. TLC of gangliosides extracted from rat organs on day 1. Gangliosides were prepared from membrane pellets as described (Materials and methods) and analysed on silica gel 60 pre-coated HPTLC plates (Merck) developed in chloroform/methanol/water containing 0.2% CaCl2, 60:40:9, v/v. Samples are pooled from organs harvested from 10 animals. Lane 1, spleen; 2, thymus; 3, small intestine; 4, colon; 5, stomach; 6, liver.
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Materials and methods Reagents The following materials were obtained from the sources indicated: [6-'H]ManNAc (20 Ci/mmol), American Radiolabelled Chemicals, St Louis, MO; Anhrobaaer ureafaaens sialidase (AUN), Calbiochem; Neu5Ac and Neu5Gc, Sigma Chemical Company; 1,2 diamino-4,5-methylenedioxybenzene, Sigma Chemical Company. Dowex 50 AG1-X2 (100-200 mesh, hydrogen form) and Dowex 3-X4A (100-200 mesh, chloride form) were purchased from BioRad, Richmond, CA. All other chemicals were of reagent gTade and were purchased from commercial sources. Animals Sprague—Dawley rats (Harlan Labs. San Diego, CA) were maintained under standard laboratory conditions. Pregnant females were maintained in the laboratory for 48 h prior to killing. For the initial study, two litters were maintained and one pup from each litter was used for each time point. For the detailed analysis, 10 pups of each age were used. Preparation of cytosolic fraction Rats of various ages were anaesthetized with inhaled methoxyfluorane and killed by decapitation. Thymus, spleen, stomach, small and large intestines, and liver were removed. Enteral contents were removed by lavage with ice-cold 50 mM Tris/HCl (pH 7.4) with 10 mM drthiothreitol. Stomach and small bowel were minced, and the mucosal layer of cokm was removed by blunt dissection. Thymus and spleen were minced, and then placed directly in the Tris/HCl buffer. Samples were homogenized by 15 x 5 s pulses with a Polytron (Brinkmann), and ultracentrifuged at 100 000 g for 1 h. Protein determination of both supernatant and pellet was performed by the Lowry method on samples dialysed against water (Lowry et aL, 1951). Supernatant was used for the assay of Neu5Ac hydroxylase and the pellet was reserved for extraction of lipids. An aliquot of the supernatant was brought up to 80% ethanol, incubated at 4°C for 15 min, and then centrifuged at 500 g for 15 min to separate free and CMP-bound sialic acids from soluble glycoproteins.
342
Extraction of total lipids Crude ganglioside extraction was performed as described previously (Ledeen and Yu, 1982). Membranes were sonicated into 19 vols of chloroform:methanol (2:1). vortexed vigorously and centrifuged at 10 000 g for 5 min. The pellet was similarly extracted sequentially with 10 ml each of chloroform: methanol (1:1) and (1:2). The combined supemates were taken to dryness. The crude lipid extract was mixed with 0.2 vols of 0.1 M KC1 and allowed to separate; the upper phase was removed. A 1:1 mixture of methanol and 0.1 M KC1 was added in a volume equal to that removed, and the procedure repeated twice. The concentrated aqueous solution was lyophilced and then dialysed against distilled water. Release of sialic acids Sialic acids were released by a two-step process. First, samples were either suspended in (gtycolipid and glycoprotein fractions) 1 ml of 100 mM sodium acetate, 4 mM calcium acetate (pH 6.0) or dialysed against this buffer (supernatants); 20 mil of sialidase from A. ureafaaens was added and the mixture incubated for 16 h at 37°C in a toluene atmosphere. After centnfugauon at 100 000 g for 30 min at 4°C, the pellet was resuspended in 2 M acetic acid and heated at 80°C for 3 h (Varki and Diaz, 1984; Diaz and Varki, 1985). The sample was again subjected to ultracentrifugation and the supernatants were pooled. The completeness of sialic acid removal was determined by spotting residual material on a TLC plate and developing with resorcinol. This was determined to be sensmve to ~ 1 nmol of sialic acid (data not shown). Purification of sialic acids Sialic acids released from glycosidic linkage by enzyme or acid hydrolysis were purified in a manner similar to that described previously (Varki and Diaz, 1984; Diaz and Varki, 1985). All steps were carried out at room temperature The supematants from the enzymatic and acid release steps were evaporated to dryness and the residue brought up in 1.0 ml of water for application to Dowex-50. The samples were loaded onto a 1 ml column of Dowex-50 (hydrogen form) The column effluent and 6 ml of water washings were collected and the pH checked. If pH > 3 , the sample was immediately passed over a 1 ml column of Dowex 3 x 4A (formate form) equilibrated in 10 mM sodium formate (pH 5 5). If pH < 3 . 100 mM sodium formate buffer (pH 5.5) was added dropwise prior to proceeding with Dowex 3 x 4A The column was then washed with 7 ml of 10 mM formic acid and the washings discarded. The sialic acids were eluted from the column with 10 ml of 1 M formic acid and the acid removed by evaporation. Analysis of sialic acids Purified sialic acids were dialysed against distilled water, concentrated and then derivatized for 2.5 h at 50°C, as described by Hara et al. (1989). HPLC was carried out using a TSK gel ODS-I20T column eluted in the isocratic mode with a mixture of acetonhnle-methanol-water (9:7:84, v/v) at a flow rate of 1.0 ml/ min at 22°C. A Linear Fluor LC 304 fluorescence detector was used at an excitation wavelength of 374 and an emission wavelength of 448. Standard assay of CMP-Neu5Ac hydroxylase Aliquots of the cytosolic fraction were added to [3H]CMP-Neu5Ac, (28.9 CM mmol) the enzyme substrate. Enzyme co-factors were added (Muchmore et al., 1989) and the mixtures were incubated at 37°C for 60 min. The reactions were stopped by the addition of 9 parts of ice-cold ethanol. The resulting precipitates were removed by centrifugation and the supematants, which contained low molecular weight cytosolic contents and CMP-sialic acids, were evaporated to dryness. The products were then directly applied to descending paper chromatography using Whatman 3mm paper with n-butanol, /i-propanol and 0.1 N HC1 (1:2:1). The papers were air-dried, cut into 1 cm fractions, soaked in distilled water and the radioactivity monitored by ^-scintillation. Enzyme activity was calculated by comparison of Neu5Ac (residual from CMP-Neu5Ac substrate) and Neu5Gc (formed by enzymatic conversion), based upon the original specific activity of the CMP-sialic acid used. Vun layer chromalography Gangliosides were analysed on silica gel 60 pre-coated HPTLC plates (Merck) developed in chloroform/methanol/water containing 0.2% CaCI,. 60:40.9, v/v. Gangliosides were visualized with resorcinol/HCl (Ledeen and Yu. 1982).
Acknowledgements The author is grateful to Monika Milewski for technical assistance The author is the recipient of a VA Career Development Award. This research was partially supported by NIGMS 1-R29 GM 43165-01.
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have activity in kidney and spleen. This raises the question of whether there may be two enzymes capable of hydrqxylating Neu5Ac, only one of which was successfully assayed in this study, or whether there are factors present in various cellular compartments which are able to modulate enzyme activity. It has been demonstrated that cytochrome b5 participates in cytosolic hydroxylation of Neu5Ac, and that antibodies directed to it abolish in vitro hydroxylation (Kozutsumi etai, 1990). Other workers have demonstrated that CMP-Neu5Ac hydroxylase activity appears to be dependent on two factors (Schneckenburger et al., 1991; Steinmetz and van Oostrum, 1991). In Sprague-Dawley rats, it is not clear whether cytochrome b 5 is involved, but Neu5Gc expression is not linearly correlated with in vitro CMP-Neu5Ac hydroxylase activity. In summary, there is a developmental sequence of sialic acid expression in both enteral and non-enteral organs of SpragueDawley rats, which appears to be distinct from environmental factors. The major sialic acids are Neu5Ac and Neu5Gc, and both exhibit a marked perinatal increase, which occurs primarily in the ganglioside fraction. Accompanying the dramatic changes in sialic acid expression on the gangliosides are shifts in the subcellular localization of sialic acids and changes in the measured activity of the CMP-Neu5Ac hydroxylase. Of note is the lack of correlation between the level of CMP-Neu5Ac hydroxylase activity and Neu5Gc expression, which implies that there is an additional level of complexity of regulation. Study of the cellular interactions dictating this developmental sequence must await the purification and genetic localization of all the enzymes involved. The Sprague—Dawley rat appears to be an excellent system in which to study the expression of Neu5Gc and its regulation because of the dramatic developmental shifts that occur.
Rat developmental sialic acid modifications
Abbreviations CMP-Neu5Ac hydroxylase, CMP-/V-acerylneuraminic acid hydroxylase; DMB, l,2-diamino-4,5-methylenedioxybenzene; ManNAc, N-acetyl-mannosamine; Neu5Ac, A'-acetylneuramink acid; Neu5Gc, W-glycolylneuraminic acid.
References
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Bouhours.D. and BouhoursJ.F. (1983) Developmental changes of hematoside of rat small intestine. Postnatal hydroxylation of fatty acids and sialic acid. J. Biol Chem.. 258, 299-304 Bouhours,D. and BouhoursJ.-F. (1988) Tissue-specific expression of Gsa (Neu5Gc) and GD3 (Neu5Gc) in epithelial cells of the small intestine of strains of inbred rats. J. Biol. Chem., 263, 15540-15545 BouhoursJ.-F. and Bouhours.D. (1989) Hydroxylation of CMP-Neu5Ac controls the expression of /V-glycolylneuraminic acid GM3 ganglioside of the small intestine of inbred rats. J. Biol. Chem., 264, 16992-16999 BouhoursJ.F., Bouhours,D. and Hansson.G.C. (1987) Developmental changes of gangliosides of the rat stomach: Appearance of a blood group B-active ganglioside. J. Biol. Chem. 262, 16370-16375. Diaz.S. and Varki,A. (1985) Metabolic labeling of sialic acids in tissue culture cell lines: methods to identify substituted and modified radioactive neuraminic acids. Anal. Biochem., 150, 32—46. Ghidoni,R. Trinchera.M., Sonnino.S , Chigorno.V. and Tettamanti.G. (1987) The sialic acid residue of exogenous GMl gangliosides is recycled for biosynthesis of sialoglycoconjugates in rat liver. Biochem. J., 247, 157—164. Gustafsson.B.E., Karlesson,K.-A., Larson.G., Midtvedt.T., Stromberg.N., Teneberg.S. and Thurin.J. (1986) Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats. J. Biol. Chem., 261, 15294-15300. Hara,S, Yamaguchi.Y., Takemori.Y., Ogura.H. and Nakamura.M. (1989) Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography Anal. Biochem., 179, 162-166. Kozutsumi.Y., Kawano.T., Yamakawa.T. and Suzuki,A. (1990) Participation of cytochrome b5 in CMP-/V-acetylneuraminic acid hydroxylation in mouse liver cytosol. J. Biochem. (Tokyo), 108, 704-706. Ledeen.R.W. and Yu.R.K. (1982) Gangliosides: Structure, Isolation, Analysis. In Ginsburg (ed ), Methods in Enzymology. Academic Press, New York, pp. 139-191. Lowry.O., Rosebrough.N., Farr,A. and Randall,R. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265. Muchmore,E.A., VarkJ.N.M., Fukuda.M. and Varki.A. (1987) Developmental regulation of sialic acid modifications in rat and human colon. FASEBJ., 1, 229-235. Muchmore,E.A., Milewski.M., Varki,A. and Diaz.S. (1989) Biosynthesis of /V-glycolylneuraminic acid: the primary site of hydroxylation of W-acetylneuraminic acid is the cytosolic sugar nucleotide pool. /. Biol. Chem., 264, 20216-20223. Schauer.R. (1982) Sialic Acids: Chemistry, Metabolism and Function. Cell Biology Monographs, Vol. 10, Springer-Verlag, New York. Schneckenburger.P., Shaw.L. and Schauer.R. (1991) Studies on the purification of CMP-W-acetylneuraminic acid hydroxylase from mouse liver. Glycoconjugate J., 8, 269. Shaw.L. and Schauer.R. (1988) The biosynthesis of ;V-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of /V-acetylneuraminic acid. Biol. Chem. Hoppe-Seyler, 369, 477^t86. Steinmetz.M. and van Oostnim,J. (1991) CMP-A'-Acetyl neuraminic acid hydroxylase activity requires at least two subcomponents Chcoconjugate J., 8, 268. Taatjes.D.J. and RothJ. (1988) Alteration in sialyltransferase and sialic acid expression accompanies cell differentiation in rat intestine. Eur. J. Cell Biol., 46, 289-298. Taatjes.D.J. and RothJ. (1990) Selective loss of sialic acid from rat small intestinal cells during postnatal development: demonstration with lectin-gold techniques. Eur. J. Cell Biol., 53, 255-266. Taatjes.D.J. and RothJ. (1991)Glycosylation in intestinal epithelium. Ini. Rev. Cytoi, 126, 135-193. Varki.A. (1992) The diversity in the sialic acids. Glycobiology, 2, 25-40. Varki.A. and Diaz.S. (1984) The release and purification of sialic acids from glycoconjugates: methods to minimize the loss and migration of 0-acetyl groups. Anal. Biochem., 137. 236-247. Received on March 20, 1992; accepted on May 17. 1992
343
Developmental sialic acid modifications in rat organs
Elaine A.Muchmore Hematology-Oncology Division, Department of Medicine, University of California at San Diego, VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, USA
Key words: A/-acetylneuraminic acid/A'-glycolylneuraminic acid/ sialic acids
Introduction Sialic acids, which are often the terminal carbohydrate moieties on cell surface glycoproteins and glycolipids, are in a unique position to have effects on cell-cell recognition. Sialic acids can be modified by the addition of various groups at the 4, 7, 8 and 9 positions. A family of naturally occurring compounds has been described which now has > 30 members (Schauer, 1982; Varki, 1992). One modified sialic acid is A/-glycolylneuraminic acid (Neu5Gc). This sialic acid is formed by conversion from the parent molecule, JV-acetylneuraminic acid (Neu5Ac), by CMP-yV-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (Shaw and Schauer, 1988; Muchmore etal., 1989). We have previously reported (Muchmore et al., 1987) that the expression of Neu5Gc in Sprague-Dawley rat colonic mucosa is developmentally regulated. It is present prenatally, disappears rapidly in the postnatal period, and then reappears. The glycoconjugates involved were not determined in this study. Several investigators have reported developmental changes on gangliosides from selected rat organs. G ^ has been determined to be the major ganglioside in rat small intestinal cells (Bouhours and Bouhours, 1983) and the sialic acid on G ^ changes from Neu5Ac at birth to Neu5Gc at the time of weaning. Furthermore, a shift from sialylation to fucosylation of rat small intestinal glycoconjugates by the weaning phase has been demonstrated (Taatjes and Roth, 1988, 1990, 1991). Developmental differences in the ganglioside composition of rat stomach mucosa have also been described (Bouhours et al., 1987). In this organ Oxford University Press
Results Neu5Gc expression is developmentally regulated in all tissues examined We have previously shown that Neu5Gc expression in rat colonic mucosal cells is developmentally regulated. In this study, sialic acids from the ganglioside fraction of multiple organs of littermates were assayed. The results in Table I are the sialic acids fractionated from the gangliosides of various organs from two littermates at each time period, expressed as nmol/mg protein in the membrane pellet. Sialic acids in the cytosol and glycoprotein pellet contribute < 10% of the total sialic acids, and are below the detectable range for many time points (data not shown). As can be seen in Figure 1, which graphically depicts sialic acids extracted from thymus gangliosides, there is a striking perinatal increase of total sialic acid/mg protein extracted from the ganglioside fraction. A similar pattern occurs in all organs sampled between days 1 and 3. No similar increase in the expression of either sialic acid was seen, although the litters were followed to day 28 (complete data not included). Although the absolute amount of Neu5Ac is greater at all time points, the increase in Neu5Gc is parallel. The majority of the sialic acid is on gangliosides Because sialic acids could not be quantitated in fractions other than the gangliosides in the time course study, a more detailed analysis of organs from 10 animals of postnatal days 1 and 6 was performed. Organs were fractionated into low molecular weight cytosolic compounds, soluble proteins, gangliosides and membrane proteins. As can be seen in Table II, >99% of the sialic acid on day 1 is present in the ganglioside fraction. Even with the larger number of animals, the amount of sialic acid in the membrane protein fraction is small in all organs. The amount of sialic acid in the ganglioside fraction decreases substantially by 337
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The changes in expression of sialic acids in Sprague-Dawley rats in the prenatal and early postnatal time period have been examined in multiple organs, both visceral and nonvisceral. In all organs examined, there is a dramatic increase in both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) shortly after birth. The bulk of the sialic acid is present in the ganglioside fraction in all tissues examined. As total amounts of sialic acid present in gangliosides decrease, the proportion present in the low molecular weight cytosolic fraction increases. A curious observation is that Neu5Ac hydroxylase activity is present at the time of the increase in sialic acid, but its activity does not correlate with Neu5Gc expression after the early postnatal period. This implies that Neu5Gc expression has another level of regulation besides CMP-Neu5Ac hydroxylase activity.
GM3 and GQ3 are the major gangliosides at birth, but GMI, G ^ , fucosyl GM1 and a blood group B-active ganglioside are added during postnatal development. In this study, the oligosaccharide portion of membrane and cytosolic structures in multiple rat organs (spleen, thymus, liver, stomach, small intestine and colon) have been examined at multiple early postnatal days, using recently described techniques for the analytical quantitation of sialic acid modifications (Hara et al., 1989). Sialic acids were localized to membrane fractions, soluble glycoproteins and low molecular weight cytosolic elements on selected postnatal days. Assays of CMP-Neu5Ac hydroxylase were carried out at all time points. The intention of the study was to determine the major oligosaccharide structures which contain the sialic acids and whether the previously seen developmental changes extended to non-visceral organs. A related question was whether Neu5Gc expression correlated with CMP-Neu5Ac hydroxylase activity.
E.A.Muchmore
Table I. Sialic acids released from gangliosides with sialidase and acetic acid, and quantified using derivatization with DMB over a TSK-ODS-120T column. The upper number for each day is Neu5Ac, the lower is New5Gc, expressed as nmot/mg protein in the organ pellet Small intestine
Day
Spleen
-1
1771 61
499 75
24 4
1
1093 93
1990 323
2
837 180 52 8
4
1.4 0.2
9
5.0 1.3
11
8.4 2.4
21
6.6 2.7
Colon
Liver
546 61
149 99
173 103
741 58
5450 263
105 14
429 22
3897 860
66 6
3244 457
416 109
23 2
43 9
128 10
406 48
507 71
156 10
n.d.
n.d
.
26 0.4
32 4.9
5.3 1.8
44 6.9
19 8.6
5.6 0.03
0.6 0.4
10 1.2
14 4.8
10 0.7
2.9 0.3
2.0 1.2
11 4.4
2.9 1.3
2.7 0.4
0.8 0.1
4.0 1.8 58 24
n.d., not determined. DMB, l,2-diamino-4,5-methylenedioxybenzene.
day 6. In this analysis, Neu5Gc is the predominant sialic acid in all organs except small intestine on day 1, implying that there is variation of Neu5Gc expression between animals. The percentage of total sialic acids in the cytosol increases on day 6 compared to day 1 As shown in Table n, the decrease in total sialic acid in the ganglioside fraction on day 6 is paralleled by an increase in total sialic acids present in the cytosol at this time. The relative increase in total sialic acids varies in each organ. The organ with the smallest change (8-fold increase) in the percentage of total sialic acids present in cytosol is the spleen, but other organs (such as the liver and colon) have a 130- to 175-fold increase between days 1 and 6 (Figure 2). The consistent trend in each organ, except the spleen, is that the predominant increase in sialic acids occurs in the ethanol supernatant. The ratio of Neu5Ac and Neu5Gc in the cytosol also varies with the organ. In all organs tested, the amount of Neu5Ac in the cytosol, in both the ethanol supernatant and pellet, is greater on day 6 compared to day 1. CMP-Neu5Ac hydroxylase activity does not correlate with Neu5Gc expression CMP-Neu5Ac hydroxylase activity is present in all organs tested in the prenatal samples (gestational day 20). After postnatal day 1, enzyme activity is consistently present in spleen and thymus, but is not detectable in small intestine or colon until day 13, and in stomach until day 28 (Figure 3). Even at day 28, activity is not detected in the liver. The level of enzyme activity does not correlate closely with Neu5Gc expressed in the ganglioside fraction. The relatively high levels in the prenatal and early postnatal period correlate with the dramatic increase in Neu5Gc 338
The ganglioside pattern changes slightly between days 1 and 6 in most organs As seen in Figures 4 and 5, both the 'a' series ( G ^ , GM2, G M h GDia. Gria) and the 'b' series (GD3, GD2, G m b , GJU,, GQ,b) are expressed at both days 1 and 6. All organs at both times have a strong, usually double, band at GM3. The chromatographic profile changes slightly in most organs between days 1 and 6. Gangliosides extracted from spleen on both days are GD|a, GD3 and GM3. In thymus, both GD3 and GM3 are seen on day 1, but only Gj^ is seen on day 6. Small intestine has Gr| b , GD3 and GM3 on day 1, but only GM3 could be seen on day 6. In colon, G Tlb , GD3 and G ^ are seen on day 1 and, on day 6, GQ]b and G D | b are also present. In stomach, the G Tlb band seen on day 1 is not seen on day 6, but on both days GD3 and GM3 are seen. In the liver, GD,a, GM1 and GM3 are seen on day 1; GD,b, GT,b appear on day 6. From this pattern, it appears that glycosyltransferases are expressed at both time points and that developmental modifications of gangliosides continue to occur. Therefore, the decrease in the total amount of ganglioside, which occurs in all organs, is not due to a difference in either the major ganglioside expressed or to a change in the developmental modifications. The sialic acids of the membrane pellet glycoproteins do not increase as those on gangliosides decrease Sialic acids released from the residual pellet after extraction with chloroform and methanol are not increased at day 6, when there is a dramatic decrease in sialic acids on gangliosides. As seen in Table I, there is not a substantial increase in either Neu5Gc or Neu5Ac on day 6 in the glycoprotein fraction in any organ. Similarly, sialic acids on glycoprotein fractions from later gestational days, when CMP-Neu5Ac hydroxylase is detectable, are not increased (data not shown).
Discussion The diversity of the sialic acids has been well established. The appreciation of the complexity of the processing has encouraged investigation regarding its biological significance. Despite the clear developmental changes noted in various organs, their biological role remains unclear (Schauer, 1982; Varki, 1992). Previous studies regarding the developmental regulation of Neu5Gc have been carried out in isolated organs of various animals (Bouhours and Bouhours, 1983, 1988, 1989; Bouhours et al., 1987; Taatjes and Roth, 1988, 1990, 1991). Many careful studies have been done on rat small intestine mucosal cells. These have showed changes in the glycoconjugates which occur both in the early postnatal period and at the time of weaning. An obvious event which could be involved in intestinal changes at these time periods is exposure to environmental organisms and their by-products. It has been demonstrated that
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7
Stomach
Thymus
during this period. However, the highest level detected is in the thymus on day 9, which is associated with only a modest rise in Neu5Gc/mg protein. In addition, the activity levels seen between days 11 and 28 are comparable to those in the prenatal and early postnatal period, but there is a clear difference in Neu5Gc expression during this period. Therefore, it appears that factors other than CMP-Neu5Ac hydroxylase activity control Neu5Gc expression.
Rat developmental sialic acid modifications
£
4000
O
o Q. 3000-
E o ea
Neu5Gc
2000 -
NeuSAc
(0 CO
1000-
perinatal
day
Fig. 1. Perinatal rat organ sialic acids. Ganglioside extracts were prepared (Materials and methods) from rat thymus from two animals at each pennatal day. Sialic acids were released with sialidase and acetic acid, and quantified using derivatization with DMB over a TSK-ODS-120T column. The open circle is Neu5Ac, the closed circle Neu5Gc, expressed as nmol/mg protein in the organ pellet. See Table II for quantification.
Table n . Sialic acids (nmol/mg protein) in cellular compartments from Sprague—Dawley rat organs on postnatal days (D) 1 and 6 Cytosol
Pellet
NGc
Ganglioside
Glycoprotein
Ethanol pellet
Ethanol soluble NAc
NGc
NAc
NGc
NAc
NGc
NAc
Spleen
Dl D6
0.5 0.6
1.6 1.2
0.09 0.7
0.05 0.99
0.05 0.3
0.22 0.7
1337 90
702 250
Thymus
Dl D6
0.5 1.2
0.04 2.9
0.44 0.13
0.02 1.4
0.08 0.4
0.16 0.6
1435 116
181 306
Small Intestine
Dl D6
0.15 1.0
0.01 18.9
0.21 0.5
0.06 2.9
0.01 0.3
0.03 0.6
28 164
90 351
Colon
Dl D6
2.0 1.5
0.16 31.2
1.0 2.5
0.19 3.1
0.2 0.02
1.1 0.1
1907 38
503 71
Stomach
Dl D6
0.63 1.09
1.62 11.4
0.15 0.14
0.06 4.8
0.33 0.06
0 27 0 26
704 79
259 174
Liver
Dl D6
0.02 0.4
0.02 0.60
0.01 0.2
0.01 0.9
0.002 0.002
0.01 0.04
24 39
29 9.9
NGc, A'-glycolylneuraminic acid. NAc, A'-acetylneuraminic acid.
the acid and non-acid glycosphingolipids of stomach, small and large intestine, and faeces of germ-free and conventional rats are chemically similar, with the exception that Neu5Gc-GM3 ' s found in the stomach of conventional, but not germ-free rats (Gustafsson et al., 1986). In the current study, sialic acid expression in various compartments of intestinal organs and spleen, thymus and liver from conventionally fed Sprague—Dawley rats has been studied. In Sprague-Dawley rats, there is a striking perinatal increase in sialic acid production in all organs tested on
days 1-2. More than 99% of the sialic acids during this period are in the ganglioside fraction. Gangliosides injected into plasma can be taken up by distant organs such as the liver (Ghidoni et al., 1987). Therefore* a concern would be that the environmental stimulus delivered to the intestinal organs stimulates ganglioside production in these organs with subsequent export of gangliosides to non-intestinal organs. Although this cannot be ruled out with certainty, the rapidity of the change in all organs tested and the observation that the gangliosides from each organ 339
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o E
E.A.Muchmore
(0
DAY 6 DAY1
30
percent Fig. 2. Percentage of total sialic acid in cytosol. Organs from 10 littermates were fractionated into low molecular weight cytosolic compounds, cytosolic proteins, membrane proteins and gangliosides (Materials and methods). The sialic acid in the cytosol is expressed as the percentage of total sialic acid. 1, spleen; 2, thymus; 3, small intestine; 4, colon; 5, stomach; 6, liver.
28 21 17 15 13
O
11
0 colon HI small bowel
CD
9
stomach
[I] thymus H spleen
7 4 2 1 -1 0.00
0.10
0.20
p m oIes/mi n/m g Fig. 3. CMP-Neu5Ac hydroxylase activity in cytosolic fractions. Cytosolic extracts from each organ were prepared and incubated with CMP-[9-3H]Neu5Ac (28.9 Ci/mmol) and co-factors as described in Materials and methods. The reactions were quenched in 90% ethanol and the sialic acids in the supernatant separated by descending paper chromatography in n-butand, n-propanol and 0.1 N HC1 (1:2:1). Radioactivity was monitored after cutting 1 cm strips. Activity is expressed in pmol/min/mg protein in the fraction.
have distinctive patterns make it unlikely. This implies that the increase in sialic acids is part of a developmental sequence that is independent of environmental factors. There is a perinatal increase in Neu5Gc which parallels the increase in Neu5Ac. In this study, the two assays of day 1 340
gangliosides demonstrated a variation in the predominant sialic acid in the gangliosides. There are several possible explanations for the differences in the proportion of Neu5Gc to total sialic acid in the early postnatal period. First, all membrane-bound sialic acids may not be accessible to release by either acid or sialidase.
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20
Rat developmental sialk add modifications
- G M3 ~ G M1 "GD3 ~GD1a
- GT1b
~
G
M3
-GD3 ~ GD1a "~GD2 -GD1b -Gjib ~ GQ1b
i Fig. 5. TLC of gangliosides extracted from rat organs on day 6. Gangliosides were prepared from membrane pellets as described (Materials and methods) and analysed on silica gel 60 pre-coated HPTLC plates (Merck) developed in chJoroform/methanol/water containing 0.2% CaCl2, 60:40:9, v/v. Samples are pooled from organs harvested from 10 animals. Lane 1, spleen; 2, thymus; 3, small intestine; 4, colon; 5, stomach; 6, liver.
Although this does not seem to be a problem with the current study because sialic acids were released with both sialidase and acid from all samples, it may have been an issue in an earlier paper (Muchmore et ai, 1987). Second is the less likely possibility that glycosyltransferases have variable expression. The feedback loops are not known, and although sialic acids do not appear to accumulate in the cytosol of perinatal specimens, this does not prove that there is no block in the formation of gangliosides. Free sialic acids in the cytosol may be degraded rapidly. Third, it is possible that the level of CMP-Neu5Ac hydroxylase activity varies between animals. Thus, if a few animals have high levels, there could be large amounts of Neu5Gc in the pool. This possibility seems the most likely because this variation would represent a subtle change in timing of the already observed decrease in CMP-Neu5Ac hydroxylase activity on day 1 in most organs. However, resolution of this issue requires analysis of individual animals, which must await more sensitive assays for the CMP-Neu5Ac hydroxylase and for the measurement of sialic acids. Between days 1 and 6, there is a change in distribution of sialic acid within the cell, such that a higher percentage is present in
both the low molecular weight cytosolic pool and in soluble proteins. The increase in sialic acid in the cytosol is primarily Neu5Ac. During this same interval the amount of sialic acid in the gangliosides decreases. The sialic acid in the cytosol may be explained by release from lysosomes of sialic acids from recycled gangliosides. It is likely that the cytosolic Neu5Gc is recycled in the intestinal organs. At day 6, there is no detectable CMP-Neu5Ac hydroxylase activity in these organs, and because this enzyme is the only known pathway for conversion of Neu5Ac to Neu5Gc, any Neu5Gc present in the cytosol would be recycled. • The lack of correlation between CMP-Neu5Ac hydroxylase activity and expression of Neu5Gc in the ganglioside fraction is unexpected. A correlation in AVN rats, which convert to GM3(Neu5Gc) in the weanling period, between CMP-Neu5Ac hydroxylase activity and CMP-Neu5Gc in the cytosol has been demonstrated (Bouhours and Bouhours, 1989), and a microsomally bound enzyme which hydroxylates intestinal glycoproteins in mutants without intestinal CMP-Neu5Ac hydroxylase activity. Of interest in Bouhours' study is the fact that AVN mutants without intestinal CMP-Neu5Ac hydroxylase activity 341
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Fig. 4. TLC of gangliosides extracted from rat organs on day 1. Gangliosides were prepared from membrane pellets as described (Materials and methods) and analysed on silica gel 60 pre-coated HPTLC plates (Merck) developed in chloroform/methanol/water containing 0.2% CaCl2, 60:40:9, v/v. Samples are pooled from organs harvested from 10 animals. Lane 1, spleen; 2, thymus; 3, small intestine; 4, colon; 5, stomach; 6, liver.
E.A.Muchmore
Materials and methods Reagents The following materials were obtained from the sources indicated: [6-'H]ManNAc (20 Ci/mmol), American Radiolabelled Chemicals, St Louis, MO; Anhrobaaer ureafaaens sialidase (AUN), Calbiochem; Neu5Ac and Neu5Gc, Sigma Chemical Company; 1,2 diamino-4,5-methylenedioxybenzene, Sigma Chemical Company. Dowex 50 AG1-X2 (100-200 mesh, hydrogen form) and Dowex 3-X4A (100-200 mesh, chloride form) were purchased from BioRad, Richmond, CA. All other chemicals were of reagent gTade and were purchased from commercial sources. Animals Sprague—Dawley rats (Harlan Labs. San Diego, CA) were maintained under standard laboratory conditions. Pregnant females were maintained in the laboratory for 48 h prior to killing. For the initial study, two litters were maintained and one pup from each litter was used for each time point. For the detailed analysis, 10 pups of each age were used. Preparation of cytosolic fraction Rats of various ages were anaesthetized with inhaled methoxyfluorane and killed by decapitation. Thymus, spleen, stomach, small and large intestines, and liver were removed. Enteral contents were removed by lavage with ice-cold 50 mM Tris/HCl (pH 7.4) with 10 mM drthiothreitol. Stomach and small bowel were minced, and the mucosal layer of cokm was removed by blunt dissection. Thymus and spleen were minced, and then placed directly in the Tris/HCl buffer. Samples were homogenized by 15 x 5 s pulses with a Polytron (Brinkmann), and ultracentrifuged at 100 000 g for 1 h. Protein determination of both supernatant and pellet was performed by the Lowry method on samples dialysed against water (Lowry et aL, 1951). Supernatant was used for the assay of Neu5Ac hydroxylase and the pellet was reserved for extraction of lipids. An aliquot of the supernatant was brought up to 80% ethanol, incubated at 4°C for 15 min, and then centrifuged at 500 g for 15 min to separate free and CMP-bound sialic acids from soluble glycoproteins.
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Extraction of total lipids Crude ganglioside extraction was performed as described previously (Ledeen and Yu, 1982). Membranes were sonicated into 19 vols of chloroform:methanol (2:1). vortexed vigorously and centrifuged at 10 000 g for 5 min. The pellet was similarly extracted sequentially with 10 ml each of chloroform: methanol (1:1) and (1:2). The combined supemates were taken to dryness. The crude lipid extract was mixed with 0.2 vols of 0.1 M KC1 and allowed to separate; the upper phase was removed. A 1:1 mixture of methanol and 0.1 M KC1 was added in a volume equal to that removed, and the procedure repeated twice. The concentrated aqueous solution was lyophilced and then dialysed against distilled water. Release of sialic acids Sialic acids were released by a two-step process. First, samples were either suspended in (gtycolipid and glycoprotein fractions) 1 ml of 100 mM sodium acetate, 4 mM calcium acetate (pH 6.0) or dialysed against this buffer (supernatants); 20 mil of sialidase from A. ureafaaens was added and the mixture incubated for 16 h at 37°C in a toluene atmosphere. After centnfugauon at 100 000 g for 30 min at 4°C, the pellet was resuspended in 2 M acetic acid and heated at 80°C for 3 h (Varki and Diaz, 1984; Diaz and Varki, 1985). The sample was again subjected to ultracentrifugation and the supernatants were pooled. The completeness of sialic acid removal was determined by spotting residual material on a TLC plate and developing with resorcinol. This was determined to be sensmve to ~ 1 nmol of sialic acid (data not shown). Purification of sialic acids Sialic acids released from glycosidic linkage by enzyme or acid hydrolysis were purified in a manner similar to that described previously (Varki and Diaz, 1984; Diaz and Varki, 1985). All steps were carried out at room temperature The supematants from the enzymatic and acid release steps were evaporated to dryness and the residue brought up in 1.0 ml of water for application to Dowex-50. The samples were loaded onto a 1 ml column of Dowex-50 (hydrogen form) The column effluent and 6 ml of water washings were collected and the pH checked. If pH > 3 , the sample was immediately passed over a 1 ml column of Dowex 3 x 4A (formate form) equilibrated in 10 mM sodium formate (pH 5 5). If pH < 3 . 100 mM sodium formate buffer (pH 5.5) was added dropwise prior to proceeding with Dowex 3 x 4A The column was then washed with 7 ml of 10 mM formic acid and the washings discarded. The sialic acids were eluted from the column with 10 ml of 1 M formic acid and the acid removed by evaporation. Analysis of sialic acids Purified sialic acids were dialysed against distilled water, concentrated and then derivatized for 2.5 h at 50°C, as described by Hara et al. (1989). HPLC was carried out using a TSK gel ODS-I20T column eluted in the isocratic mode with a mixture of acetonhnle-methanol-water (9:7:84, v/v) at a flow rate of 1.0 ml/ min at 22°C. A Linear Fluor LC 304 fluorescence detector was used at an excitation wavelength of 374 and an emission wavelength of 448. Standard assay of CMP-Neu5Ac hydroxylase Aliquots of the cytosolic fraction were added to [3H]CMP-Neu5Ac, (28.9 CM mmol) the enzyme substrate. Enzyme co-factors were added (Muchmore et al., 1989) and the mixtures were incubated at 37°C for 60 min. The reactions were stopped by the addition of 9 parts of ice-cold ethanol. The resulting precipitates were removed by centrifugation and the supematants, which contained low molecular weight cytosolic contents and CMP-sialic acids, were evaporated to dryness. The products were then directly applied to descending paper chromatography using Whatman 3mm paper with n-butanol, /i-propanol and 0.1 N HC1 (1:2:1). The papers were air-dried, cut into 1 cm fractions, soaked in distilled water and the radioactivity monitored by ^-scintillation. Enzyme activity was calculated by comparison of Neu5Ac (residual from CMP-Neu5Ac substrate) and Neu5Gc (formed by enzymatic conversion), based upon the original specific activity of the CMP-sialic acid used. Vun layer chromalography Gangliosides were analysed on silica gel 60 pre-coated HPTLC plates (Merck) developed in chloroform/methanol/water containing 0.2% CaCI,. 60:40.9, v/v. Gangliosides were visualized with resorcinol/HCl (Ledeen and Yu. 1982).
Acknowledgements The author is grateful to Monika Milewski for technical assistance The author is the recipient of a VA Career Development Award. This research was partially supported by NIGMS 1-R29 GM 43165-01.
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have activity in kidney and spleen. This raises the question of whether there may be two enzymes capable of hydrqxylating Neu5Ac, only one of which was successfully assayed in this study, or whether there are factors present in various cellular compartments which are able to modulate enzyme activity. It has been demonstrated that cytochrome b5 participates in cytosolic hydroxylation of Neu5Ac, and that antibodies directed to it abolish in vitro hydroxylation (Kozutsumi etai, 1990). Other workers have demonstrated that CMP-Neu5Ac hydroxylase activity appears to be dependent on two factors (Schneckenburger et al., 1991; Steinmetz and van Oostrum, 1991). In Sprague-Dawley rats, it is not clear whether cytochrome b 5 is involved, but Neu5Gc expression is not linearly correlated with in vitro CMP-Neu5Ac hydroxylase activity. In summary, there is a developmental sequence of sialic acid expression in both enteral and non-enteral organs of SpragueDawley rats, which appears to be distinct from environmental factors. The major sialic acids are Neu5Ac and Neu5Gc, and both exhibit a marked perinatal increase, which occurs primarily in the ganglioside fraction. Accompanying the dramatic changes in sialic acid expression on the gangliosides are shifts in the subcellular localization of sialic acids and changes in the measured activity of the CMP-Neu5Ac hydroxylase. Of note is the lack of correlation between the level of CMP-Neu5Ac hydroxylase activity and Neu5Gc expression, which implies that there is an additional level of complexity of regulation. Study of the cellular interactions dictating this developmental sequence must await the purification and genetic localization of all the enzymes involved. The Sprague—Dawley rat appears to be an excellent system in which to study the expression of Neu5Gc and its regulation because of the dramatic developmental shifts that occur.
Rat developmental sialic acid modifications
Abbreviations CMP-Neu5Ac hydroxylase, CMP-/V-acerylneuraminic acid hydroxylase; DMB, l,2-diamino-4,5-methylenedioxybenzene; ManNAc, N-acetyl-mannosamine; Neu5Ac, A'-acetylneuramink acid; Neu5Gc, W-glycolylneuraminic acid.
References
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