Sialic acids are a family of more than 40 different 9-carbon carboxylated 2-keto sugars (Schauer et al., 1995) found in most living organisms ranging from prokaryotes to higher animals with the exception of certain bacteria. They often occur in exposed peripheral positions of the oligosaccharide moiety of glycoconjugates, thus appearing as likely candidates for recognition determinants (Kelm and Schauer, 1997). In addition, some sialic acids are developmentally regulated and have been shown to be re-expressed in a variety of malignant tumors, thereby acting as oncodevelopmental markers (Narayanan, 1994). Overall, besides their expected contribution to the physicochemical properties of glycoconjugates and cells, primarily derived from their strong negative charge, sialic acids play a central role in many crucial recognition processes, such as trafficking of proteins and cells, bacterial and viral infection, development, and tumor progression (Reutter et al., 1997).
The most commonly occurring sialic acids are N-acetylneuraminic acid (Neu5Ac) and N-glycoloylneuraminic acid (Neu5Gc). Structural diversity arises from various substitutions (e.g., O-acetylation) at positions 4-7-8-9. A unique deaminated sialic acid (3-deoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid, Kdn) was initially discovered in rainbow trout eggs (Nadano et al., 1986) and lately found in different bacteria, lower vertebrates (Knirel et al., 1989; Strecker et al., 1992) and higher vertebrates including mammals (Ziak et al., 1996). Kdn has been detected and quantitated in several tissues from different mammals by fluorometric high-performance liquid chromatography after derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) (Inoue et al., 1996). Although this method has been extensively used for analysis of sialic acids (Hara et al., 1987, 1989), the response of Kdn to derivatization with DMB has not been so far reported.
In this work we report on the content and subcellular distribution of Kdn in rat liver, an organ which had not yet been studied. Optimal conditions for derivatization of Kdn with DMB and quantitation by fluorometric reverse-phase HPLC have been previously established. Expression of rat liver Kdn, Neu5Gc, and Neu5Ac changes significantly and independently with postnatal development and aging.
Studies on the derivatization of Kdn with DMB
DMB reacts with [alpha]-keto acids to produce quinoxalinone fluorescent derivatives (Nakamura et al., 1987) that can be separated by reverse-phase HPLC (Hara et al., 1987, 1989). The influence of temperature and time of reaction on the formation of the quinoxalinones of Neu5Gc, Neu5Ac, and some of their O-acetylated derivatives had been studied by Hara et al. (1989). However, no data were available on the derivatization of Kdn. Therefore, we studied the kinetics of the reaction of Kdn with DMB at different temperatures.
At 50°C, which is the optimum temperature previously reported for derivatization of Neu5Ac and Neu5Gc (Hara et al., 1989), the reaction of DMB with Kdn increased almost linearly with time and appeared to reach a plateau after 5-6 h of incubation. This behavior differed from that exhibited by Neu5Ac and Neu5Gc for which derivatization was almost complete after 1 h (Figure
Figure 1. Kinetics of derivatization with DMB of Kdn (solid triangles), Neu5Gc (solid squares), and Neu5Ac (solid circles) at different temperatures. Derivatization was carried out for different intervals of time at 50°C (A), 65°C (B), and 80°C (C); 10 µl of the reaction mixture, containing about 80 pmol of each sialic acid, was applied to the column and eluted isocratically with acetonitrile/methanol/water (9:7:84).
The corrected fluorescence excitation and emission spectra of the Kdn quinoxalinone (not shown) were identical to those of the Neu5Ac derivative (excitation maximum, 370 nm; emission maximum, 448 nm) and very similar to those reported for several different [alpha]-ketoacids (Nakamura et al., 1987).
Detection of Kdn in rat liver homogenates
The presence of Kdn in rat liver was investigated using different populations of defined age. The results obtained within each group were highly reproducible.
The elution position of the quinoxalinone fluorescent derivatives of Kdn, Neu5Gc, and Neu5Ac was first determined using standard sialic acids (Figure
Figure 2. HPLC elution profiles of the DMB-derivatized standard sialic acids (A) and sialic acids derived from adult (B) and old rat (C) liver homogenates. Sialic acids were released with 2 M acetic acid at 80°C for 5 h, purified by ion-exchange chromatography and derivatized with DMB at 50°C for 5 h. Elution was performed under the concave gradient mode as specified in Materials and methods. Peaks: 1, Kdn; 2, Neu5Gc; and 3, Neu5Ac.
Table I.
Age
Neu5Ac
Neu5Gc
Kdn
pmol/mg protein
Rel. amount
%
pmol/mg protein
Rel. amount
%
pmol/mg protein
Rel. amount
%
Newborn
2000 ± 212
1.0
83.3
370 ± 38
1.0
15.4
30 ± 3.8
1.0
1.3
7 days
1800
0.9
89.1
200
0.54
9.9
20
0.67
1.0
4-6 months
1100 ± 140
0.55
95.4
50 ± 5.5
0.13
4.3
3 ± 0.5
0.1
0.3
24 months
634 ± 70
0.32
92.8
36 ± 4
0.10
5.3
13 ± 2
0.43
1.9
Table II.
Age | Sialic acid | Membrane-associated glycoconjugates | Cytosolic fraction | ||
Newborn | Neu5Ac | 63-53b | 37-47b | ||
Neu5Gc | 90-85 | 10-15 | |||
Kdn | 2 | 98 | |||
Glycoproteins | Glycolipids | Glycoproteins | Free and CMP-bound | ||
4-6 months | Neu5Ac | 52.8 | 30.8 | 10.2 | 6.2 |
Neu5Gc | 54.4 | 37 | 8.6 | n.d.c | |
Kdn | <10 | n.d. | n.d. | >90 | |
24 months | Neu5Ac | 42.4 | 7.8 | 38.5 | 11.5 |
Neu5Gc | 50 | 14.1 | 35.3 | 0.7 | |
Kdn | 3.8 | 6.2 | n.d. | 90 |
Subcellular distribution of Kdn, Neu5Gc, and Neu5Ac
Rat liver homogenates were subjected to differential centrifugation and the amount of Kdn, Neu5Gc, and Neu5Ac in the different fractions was estimated after HPLC separation. In adult rats most of the Kdn contained in the homogenates was present in the soluble cytoplasmic fraction, while Neu5Ac and Neu5Gc were mainly associated with membrane-bound glycoconjugates (Figure
Figure 3. Distribution of Kdn, Neu5Gc, and Neu5Ac in the different subcellular fractions of adult rat liver homogenates. The amount of sialic acids is expressed as nmol per gram of processed tissue.
In order to identify the nature of the molecules to which Kdn was bound, rat liver homogenates were fractionated into the membrane-associated glycoconjugates and the cytosolic soluble components (Table II).
Only less than 10% of Kdn in rat liver homogenates was associated to membrane glycoproteins, while more than 90% was present in the cytosol as free and/or CMP-bound Kdn since it could be completely extracted from the cytosolic fraction with cold ethanol. Most of the cytosolic Kdn was unaffected by treatment with sodium borohydride, while more than 90% of standard Kdn was reduced in the same conditions (data not shown), suggesting that Kdn is present in the cytosol mostly in the form of the nucleotide precursor.
No significant variations in the relative proportion of cytosolic Kdn were observed with age. However, there were some differences in Kdn associated to membrane glycoconjugates. Thus, in old animals a small percentage could be detected in membrane glycolipids, while in newborn and adult rats no detectable amounts were present in this fraction. On the contrary, Neu5Ac and, in particular, Neu5Gc were found to be mainly associated to membrane glycoconjugates.
In old animals the amount of both Neu5Ac and Neu5G bound to membrane glycolipids decreased considerably (from 30.8 and 37% to 7.8 and 14.1%, respectively), and this decrease was paralleled by an increase in the amount present in soluble glycoproteins and, to a minor extent, in the ethanol extracts.
The studies here described indicate that rat liver contains age-regulated Kdn which is present fundamentally in the cytosolic fraction as the nucleotide precursor.
Kdn has been determined by fluorometric reverse-phase HPLC of the corresponding quinoxalinone, a method that has already been applied to the detection and quantitation of Kdn in other rat tissues (Inoue et al.,1996). However, no details on the response of Kdn to derivatization with DMB has been reported. Here we show that the kinetics of derivatization of Kdn is slower than those of Neu5Ac and Neu5Gc, whereas the fluorescence intensity of the Kdn quinoxalinone product is 2.5-fold higher than those of the Neu5Gc and Neu5Ac derivatives. This different response of Kdn to derivatization has been taken into account for calculating the amount of this sialic acid present in biological samples. In addition, detection and quantitation of Kdn in the HPLC chromatograms has been facilitated by using a concave gradient elution which increases the separation of Kdn and Neu5Gc from 0.4 min, obtained under isocratic elution, to 3 min. The amount of Kdn, Neu5Gc, and Neu5Ac thus found in adult rat liver was 3, 50, and 1100 pmol per mg of protein, respectively. The relative proportion of Kdn is only 0.3%, which is comparable to that we observed in other organs such as spleen or lung and clearly smaller than that found in submaxillary gland (unpublished observations) in agreement with the results reported by Inoue et al. (1996).
Most of Kdn in rat liver is associated with the cytosolic fraction as nucleotide precursor, as can be deduced by the fact that it could be extracted with cold ethanol and was unmodified after treatment with sodium borohydride. The occurrence of cytosolic CMP-sialic acids is well established (Bouhours and Bouhours, 1989; Muchmore et al., 1989). According to the present knowledge, CMP-Neu5Ac is synthesized in the cell nucleus and diffuses into the cytosol (Coates et al., 1980). There, it can be hydroxylated to CMP-NeuGc by a CMP-NeuAc hydroxylase (Bouhours and Bouhours, 1989). Both types of CMP-sialic acids are transported into Golgi vesicles to be available for sialyltransferases. The mechanism of incorporation of Kdn to glycoconjugates is still unknown, although a CMP-Kdn synthetase (Terada et al., 1993) and a Kdn-transferase (Angata et al., 1994) have been identified and characterized in rainbow trout.
Also, recently it has been found that rat liver Gal [beta]1->4 GlcNAc [alpha]2,6-sialyltransferase is able to catalyze the transfer of Kdn from CMP-Kdn to acceptor glycoconjugates with as much as half the efficiency of that of Neu5Ac from CMP-Neu5Ac (Angata et al., 1998). However, even if a specific Kdn-transferase was present in rat liver, the concentrations of CMP-Kdn could be too low to allow an effective activity of the sialyltransferases, thus explaining the small percentage of Kdn incorporated to glycoconjugates.
The expression of Kdn, Neu5Gc, and Neu5Ac in rat liver decreases significantly with postnatal development from newborn to adult rats. The composition of the oligosaccharide moiety of glycoconjugates is known to be altered during development and differentiation (Fenderson et al., 1990). Sialic acids, because of their predominantly terminal position within the oligosaccharide chain, their electronegative charge at physiological pH, and their remarkable structural variability, are known to be actively involved in cell-cell recognition events and therefore to be developmentally regulated. In fact, variations in the total amount of sialic acids and in the expression of the different species during development has been extensively reported. The decrease in Neu5Gc and Neu5Ac expression in rat liver from newborn to adult rats, reported in this work, is in agreement with the results obtained with one of the systems more extensively studied: the polysialylated neural cell adhesion molecule (PSA-NCAM). Thus, the molecule present in adult chicken brain has been reported to contain only 10% sialic acid as opposed to 30% sialic acid in embryonic NCAM (Rothbard et al., 1982). This difference has been correlated to changes in the expression of sialyltransferases with development (Phillips et al., 1997). Also the sialic acid content of small intestinal epithelial cells from rats (Taatjes and Roth, 1990) and pigs (King et al., 1995) has been found to be highest in newborns decreasing through weaning and reaching lowest levels in adults. Concerning Kdn, Inoue et al. (1996) reported a higher content in some tissues as neocortex, olfatory bulb, and lung of 7-day-old rats as compared with adults, while the opposite was found in heart. Here we show that the expression of rat liver Kdn is also higher in newborn and 7-day-old rats, than in adults.
Less work has been done related to variations in sialic acids with aging. We have found a decrease in the amount of Neu5Ac and Neu5Gc in old rats, which is similar to the decrease in PSA-NCAM in aged rats (Fox et al., 1995), in the sialylation of human IgG (Keusch et al., 1996), and in human brain gangliosides (Svennerholm et al., 1997) reported by others. On the contrary, Kdn content was higher in aged rats than in adults. This increment could be related to a normal process of aging or to the presence of tumors that very often occurs in aged rats. Actually human lung oligo/poly-Kdn has been found to be re-expressed in various histological types of lung carcinomas, representing a new oncodevelopmental antigen in lung (Qu et al., 1996).
The distribution of Kdn between the different sialic containing molecules does not change significantly with age: the percentage found as nucleotide precursor was always around 90%. However, the distribution of Neu5Ac and Neu5Gc changes considerably with aging. In particular, there is an important decrease in Neu5Ac and Neu5Gc bound to membrane glycolipids that correlates with an increase in cytosolic components, mainly soluble glycoproteins.
As a whole, the variations in the expression of Kdn, Neu5Gc, and Neu5Ac with age do not follow a single pattern, suggesting that these sialic acids are independently regulated. The biological significance of this regulation remains to be elucidated.
Chemicals
Neu5Ac, Neu5Gc, DMB, and [beta]-mercaptoethanol were purchased from Sigma (St. Louis, MO). Kdn was from Toronto Research Chemicals Inc. Sodium hydrosulfite and solvents of HPLC grade were from Merck.
Animals
Male Wistar rats were provided by the Instituto Cajal, C.S.I.C., Madrid. The amount of sialic acids present in rat liver was studied in four age populations. (1) Newborn: two different preparations, each from four newborn rats, were studied. (2) Seven days old: only one preparation from two rats. (3)Adult: three preparations, each from a single rat aging from 4 to 6 months. (4) Aged: two preparations, each from a single rat aging 22-24 months.
Subcellular fractionation of liver homogenates
Rats were decapitated and the liver removed, immediately frozen and kept at -70°C until use. Livers were minced; washed with 10 mM Tris-HCl, pH 7.2, containing 280 mM sucrose and 0.5 mM EDTA; and homogenized with a Potter-Elvehjem in the same buffer (10 ml per g of liver). The homogenate was spun down at 650 gmax for 10 min, and a nuclear pellet and a postnuclear supernatant were collected. This first supernatant was then centrifuged at 10,400 gmax for 8 min to obtain a pellet enriched in mitochondria and a second supernatant which was further centrifuged at 100,000 gmax for 1 h to yield a pellet of microsomes and a third supernatant containing the soluble components of the cytoplasm. No further purification of the different fractions was attempted.
Gangliosides were extracted with chloroform/methanol/water (4:8:3, v/v) using the optimized method described by Schnaar (1994). Free and CMP-bound sialic acids from soluble cytosolic fractions were extracted with ice-cold ethanol (80% final concentration) as described by Bouhours and Bouhours (1989). Aliquots of these extracts were treated with sodium borohydride to differentiate CMP-bound from free sialic acids (Seppala et al. 1991). Control experiments were carried out in parallel with standard sialic acids.
Release and purification of sialic acids
Sialic acids were released by treatment with acetic acid (2 M final concentration) at 80°C. Different incubation times were previously tested to get maximum release of sialic acids. It was observed that completion of the hydrolysis depended on the different cellular fractions studied. However, after 5 h of incubation with acetic acid release of sialic acids had reached a plateau for all the samples. Further incubation resulted in degradation of released sialic acids. The course of the hydrolysis was also affected by the concentration of the sample. Protein concentrations were always kept below 10 mg protein/ml, since above this value an impairment in the release of sialic acids was observed. The hydrolyzates were centrifuged for 10 min at 13,000 r.p.m. in a bench microfuge and the supernatants were purified by ion exchange chromatography on Dowex-1 (X8, 200-400 mesh, formate form).
Derivatization
An aliquot of the sample solution was mixed with an equal volume of 7 mM DMB in 1.4 M acetic acid, containing 1 M [beta]-mercaptoethanol and 18 mM sodium hydrosulfite (Hara et al., 1989) and heated for 5 h at 50°C, unless otherwise stated.
HPLC apparatus and conditions
A Shimadzu LC-4A high-performance liquid chromatograph equipped with a Rheodyne 7125 syringe-loading sample injector valve and a Waters 420 fluorescence detector (set at wavelengths of 338 nm excitation and 425 nm emission) was used. Chromatographic data were recorded using a Shimadzu C-R3A Chromatopac data processor. The column was a Hypersil ODS C18, particle size 5 µm (Sugelabor, Madrid, Spain). Chromatography was carried out at room temperature and at a flow rate of 1 ml/min. Where specified, elution was performed isocratically with acetonitrile/methanol/water (9:7:84) as described by Hara et al. (1989). However, under these conditions the difference in the retention times between the DMB derivatives of Kdn and Neu5Gc was 0.3 min, resulting in only marginal resolution of both sialic acids. Therefore, different elution modes and mobile-phase compositions were consecutively assayed in order to optimize the separation of these derivatives. The best resolution was obtained using a concave gradient from 11 to 50% of (acetonitrile/methanol,57:43):water during 30 min. The concavity of the gradient was determined by a curvature parameter c = 5, according to the equation:
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where T is the total gradient time. Under these conditions the difference in the elution times between the derivatives of Kdn and Neu5Gc was 3 min.
Fluorescence spectra
Fluorescence excitation and emission spectra of eluates of the peaks corresponding to Kdn and Neu5Ac quinoxalinones were measured in a SLM 8000D spectrofluorimeter; the spectral bandwidths were 2 nm in both the excitation and emission monochromators.
We gratefully acknowledge Dr. Pilar Lillo for the fluorescence spectra, Drs. J.C.Díez Masas and M.Frutos for many helpful discussions, Mrs. Mª Luisa Ruiz Pineda for technical assistance and Mrs. Nieves Salvador for provision of the rat livers used in these studies. This work was supported by Grant SAF 92-0497 from Dirección General de Investigación Científica and Grants 93-0317 and 90-0014 from Fondo de Investigaciones Sanitarias.
Kdn, 3-deoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycoloylneuraminic acid; DMB, 1,2-diamino-4,5-methylenedioxybenzene; PSA-NCAM, polysialylated neural cell adhesion molecule.
3To whom correspondence should be addressed at: Instituto de Química Física "Rocasolano," Serrano 119, 28006 Madrid, Spain