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Keywords:

  • membrane-associated;
  • Neu2;
  • plasma membrane;
  • thymus

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4MU-Neu5Ac as a substrate. The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP-40 or DOC/NP-40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same pI as that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris-buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane-rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2-transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.

List of Abbreviations
C

chloroform

D

DOC/NP-40-solubilized fraction

DOC

deoxycholate

DN

CD4CD8 double negative

DP

double positive

IEF

isoelectric focusing

lpr

lymphoproliferation

M

methanol

4MU-Neu5Ac

4-methylumbelliferyl 5-acetyl neuraminic acid

N

NP-40-solubilized fraction

NP-40

nonidet P-40

pI

isoelectric point

PNA

peanut agglutinin

S

soluble fraction

SP

single positive.

Cell-to-cell interactions are important for the exchange of information among cells, especially in the immune system. The removal of sialic acid from the cell surface strongly affects this interaction. We have previously reported that sialidase treatment of Epstein–Barr virus (EBV)-transformed B cells resulted in large cell aggregation [1, 2]. We speculated that a natural sialidase might exist on the surfaces of immune cells and searched for a membrane-associated sialidase that acts at a neutral pH in immune organs. We found a high level of sialidase activity at a neutral pH in the mouse thymus not only in the soluble fraction [3] but also in the crude membrane fraction [4]. We also determined that a unique cell, a ‘Neu-medullocyte’ (Mac-1 and immunoglobulin-positive cell), exists in the thymus of Neu1b and Neu1c mice [4, 5], whereas this cell and soluble Neu2 sialidase activity are absent in the SM/J (Neu1a) mice [5]. The SM/J mouse is a naturally occurring strain that exhibits a partial deficiency in neuraminidase [6] due to a point mutation in the Neu1 locus [7, 8]. We sequenced the Neu2 mRNA from the A/J (Neu1b) mouse [3], discovered a novel sequence at the 5′ terminus (Neu2B) compared to the muscle Neu2 (Neu2C) [9], and studied several characteristics of these sialidases [10].

However, the membranous sialidase that acts at neutral pH in the thymus remains unidentified. The sialidases that have been cloned from vertebrates include four kinds (Neu1, 2, 3, and 4), and comprehensive reviews have been published concerning these properties [11, 12]. Neu1, 3, and 4 have optimal activity at acidic pH whereas Neu2 is optimal at neutral pH. The neutral activity in the thymus crude membrane cannot be explained by these acidic sialidase activities.

The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane of the thymus. We attempted to clarify the characteristics of the sialidase and determined that a plasma membrane-rich fraction from the mouse thymus exhibited a sialidase activity that removed sialic acid from fetuin at pH 7.0 but not at pH 4.5. We concluded that the molecule that is primarily responsible for the main activity at neutral pH in the crude membrane fraction is most likely the Neu2 molecule, which is associated with the membranous fraction by an unknown mechanism.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Chemicals and sialidase substrates

The following materials were obtained commercially: 4MU-Neu5Ac was purchased from Nacalai Tesque (Kyoto, Japan); fetuin, sialyllactose, mucin, and a ganglioside mixture from the bovine brain (type II) were obtained from Sigma (St Louis, MO, USA). PNA was obtained from Seikagaku Kogyo, Co., Ltd (Tokyo, Japan), biotin-labeled lectins were obtained from J-OIL MILLS, Inc. (Tokyo, Japan), and Ampholine was obtained from LKB (Stockholm, Sweden). Total gangliosides for the natural substrate of sialidases were prepared from 18 g pooled murine spleens. Briefly, total lipids were extracted with 200 mL each of chloroform (C)/methanol (M) (2:1) and C/M/water (W) (1:2:0.8). The total ganglioside fraction was obtained using DEAE-Sephadex and eluted with C/M/0.8 M sodium acetate and condensed after dialysis. We detected two purple spots by resorcinol reagent in the middle and lower parts of the thin layer chromatography (TLC) developed with C/M/0.2% CaCl2 aq. (55:45:10). The sialic acid content was quantified by the thiobarbituric acid method. The crude membrane fraction was prepared from the mouse thymuses for use as an endogenous substrate as described below and was divided into small tubes and frozen until use.

Mice, antibodies, and transfection

A/J and C57BL/6 mice were purchased from Shizuoka Laboratory Animal Center Hamamatsu, Shizuoka, Japan. MRL/lpr, MRL/n, B6/lpr, and B6/n mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). SMXA recombinant mice were kindly donated through Hamamatsu Medical School and Nagoya University (Japan) by Dr M. Nishimura, who established these mice when he was at Hamamatsu Medical School [13]. After the mice were killed, the thymus, spleen, and peripheral lymph nodes were carefully dissected and were either used immediately or frozen at –70°C until use.

PE-labeled anti-CD8a (BD Biosciences, San Jose, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (CALTAG Laboratories, Bangkok, Thailand) were obtained commercially. Anti-Neu2 rabbit polyclonal antibody was produced by immunizing a synthetic peptide corresponding to the Glu39-Ser58 segment of the rat cytosolic Neu2 [9], and the antisera preparation was purified using an affinity column coupled with the synthetic peptide. Commercial anti-Neu2 (M-13) sc-168736 goat polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit non-immunized serum was prepared in our laboratory. COS-1 cells were transfected with Neu1, Neu2C, Neu3, Neu4S, or Neu4L cDNA, and the homogenates were prepared and used as their respective enzyme sources. COS-7 cells transfected with the Neu2B plasmid or the Neu2C plasmid were prepared as described previously [10].

Measurement of sialidase activity

Sialidase activity using 4MU-Neu5Ac as a substrate was assayed by the spectrofluorometric measurement of released 4MU in a large-scale method with a final volume of 1 mL (Hitachi spectrofluorometer, Tokyo, Japan) [4] or in a small-scale method with a final volume of 0.2 mL using a SpectraMax M5 multimode micro-plate reader (Molecular Devices Japan, Tokyo, Japan) [10]. With substrates other than 4MU-Neu5Ac, the amount of released sialic acid was determined by the thiobarbituric acid method [14] or by fluorometric high-performance liquid chromatography with malononitrile [15].

SDS-PAGE and Western blot analysis

NP40-solubilized protein from the crude membrane fraction from normal or lpr thymus was applied for SDS-PAGE under reducing conditions with 10% acryl amide gel with the size marker (APRO Science, Naruto, Tokushima, Japan) and transferred to a polyvinylidene fluoride (PVDF) membrane (Hybond-P; Amersham Pharmacia Biotech, Little Chalfont, UK) [16]. A sheet was treated with 0.1 N trifluoroacetic acid at 65°C for 2 hr [17] to remove sialic acid and all membranes were subsequently blocked with 1% BSA in Tris-Tween-saline (TTS, 10 mM Tris-HCl, pH 7.5/0.05% Tween/0.15 M NaCl). After washing with TTS, the blot was stained with biotin-labeled lectin and StAv-horseradish peroxidase. Enhanced chemiluminescence (ECL) generated with ECL plus reagent was detected using X-ray film.

Extraction and analysis of ganglioside from thymus

The frozen thymuses (500 mg wet weight) were homogenized and lipids were extracted with C/M by an orthodox method and applied to DEAE column chromatography. After the alkaline methanolysis of the acid glycolipid fraction, gangliosides were separated by TLC with C/M/0.5% CaCl2 aq. (55:45:10) solution and observed with resorcinol reagent. The residue after C/M extraction was solubilized by alkaline treatment, and the protein amounts were determined.

Preparation of the thymus homogenate

The thymus homogenates were prepared according to the method described previously [4]. Briefly, frozen or fresh thymuses (6- to 9-week-old mice) were homogenized with a Dounce homogenizer using 100 volumes of 1 mM NaHCO3/0.1 mM CaCl2. After 5 min of slow mixing, the filtrate that had been passed through three layers of nylon mesh was used as the total homogenate and was centrifuged at 450g for 10 min; next the supernatant was used as the thymus homogenate.

Preparation of the S, N, and D fractions from the thymus homogenate

The thymus homogenate described above was centrifuged at 105,000g for 1 hr, and the soluble (S) fraction and the precipitate were obtained. The precipitate was designated the crude membrane fraction. The crude membrane fraction was solubilized using sonication buffer, DNase, and NP-40-solubilizing buffer (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl containing 0.5% NP-40, 200 µg phenylmethylsulfonyl fluoride/mL) [18] on ice with stirring for 30 min. After centrifugation at 90,000g for 30 min, the supernatant was designated the N fraction. The insoluble precipitate was solubilized again using NP40/DOC solubilizing buffer (2.5 mM DOC in the NP-40-solubilizing buffer), and the solubilized fraction after the centrifugation described above was designated the D fraction.

Subcellular fractionation of the thymus crude membrane

The crude membrane fraction described above was suspended in PBS by sonication, overlaid on 35% sucrose in PBS and centrifuged at 24,000g for 1 hr using a Hitachi swing rotor SW28 (Hitachi, Tokyo, Japan). Four fractions, the upper, middle, lower, and precipitate fractions, were separated and mixed with PBS and centrifuged at 105,000g for 1 hr. Each precipitate was then suspended in PBS and sialidase activity was assayed with 4MU-Neu5Ac at pH 7.0 and pH 4.5 in citrate-phosphate buffer. The sialic acid released from the other substrate was assayed by the thiobarbituric acid method [14].

Washing of the crude membrane

The crude membrane fraction was washed according to the method described previously [10]. The sialidase activity in the precipitate and the supernatant fractions was determined using 4MU-Neu5Ac as a substrate.

Preparative isoelectric focusing

The S, N, and D fractions prepared from pooled frozen thymuses described above were individually added to a solution containing 0.8 mL Ampholine pH 3 − 10 and 0.4 mL each of Ampholine pH 4 − 6, pH 6 − 8, and pH 8 − 9.5. The volume of each sample was adjusted to 40 mL with water and subjected to IEF using a Rotofor cell system (BioRad, Hercules, CA, USA) at 4°C for 4 − 4.5 hr at 12 W with 1 M H3PO4 as the anode solution and 1 M NaOH as the cathode solution. The solution in the Rotofor cell was separated into 20 tubes, the pH was measured and the protein concentration was determined using the Bio-Rad Protein Assay Kit. The first five and last four fractions were dialyzed against 10 mM Tris buffer (pH 7.4) for 4 hr to remove H3PO4 or NaOH, concentrated using dialysis tubing covered with dried Sephadex beads (Pharmacia Fine Chemicals, Uppsala, Sweden), and dialyzed against PBS for 30 min before assaying for sialidase activity.

Preparation of T cells, non-T cells, PNA-aggregated and PNA-unaggregated T cells

The freshly removed thymus was gently crushed by hand using a loose frosted glass homogenizer in RPMI medium and the cell suspension was passed through three layers of nylon mesh. The passed cells were denominated T cells. The cells remaining on the nylon mesh were designated non-T cells. The T cells were divided into PNA-aggregated and PNA-unaggregated T cells by the PNA method as described previously [5, 19]. PNA-aggregated cells consisted of immature T (CD4+CD8+ DP) cells and PNA-unaggregated T cells consisted of mature T (CD4+ SP and CD8+ SP) cells and immature CD4CD8 DN cells [20].

T-cell sorting into DN, DP, CD4+ SP, and CD8+ SP cells

T cells prepared from the four thymuses of C57BL/6 mice (6 weeks old) were labeled with FITC-conjugated anti-mouse CD4 and phycoerythrin (PE)-labeled anti-CD8a in 1.5 mL PBS/0.1% BSA/0.1% NaN3 for 30 min on ice and washed two times in PBS, suspended in 2 mL PBS/0.1% BSA/0.1% NaN3, and then sorted into DN, DP, CD4+ SP, and CD8+ SP cells using a BD FACSAria™ II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Sialidase activity level in various organs with age

Figure 1 shows the level of sialidase activity with 4MU-Neu5Ac in the various organs and age. The sialidase activity level in the thymus at pH 7.0 was the highest among the organs tested for activity in both of the soluble and crude membrane fractions (Fig. 1a). Although the weight of the thymus did not decrease at 17 weeks (Fig. 1b), the activity level in the soluble fraction at pH 7.0 decreased to less than half of the maximum at 17 weeks (Fig. 1c): levels in the crude membrane fraction at pH 7.0 and 4.5 did not decrease. The spleen and lymph node exhibited little activity (Fig. 1c). Thus, the high levels of sialidase activity at pH 7.0 observed in the soluble and crude membrane fractions are unique in the thymus, and the decrease in the activity level at approximately 17 weeks is unique in the soluble fraction of the thymus.

image

Figure 1. Sialidase activity levels in various organs and their activity levels with age. (a) Sialidase activity level toward 4MU-Neu5Ac was measured in various tissues from a 9-week-old A/J mouse. Each tissue homogenate was fractionated into soluble (Sol) and crude membrane (CM) fractions by centrifugation (105,000g, 30 min). Each value represents the average of two assays. (b) Relationship between the weight of the thymus and age (number of weeks) in A/J mice. (c) Sialidase activity levels in the thymus (Thy), lymph node (LN), and spleen (Spl) from A/J mice at various ages. Left and right figure panels show the activity level of the Sol and the CM fraction, respectively. Each value represents the average of two assays.

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Natural substrates for the sialidases in the S, N, and D fractions obtained from the thymus homogenate

We obtained the S, N, and D fractions from the thymus homogenate and investigated the activity level of sialidase with five substrates (Table 1). The N fraction exhibited activity with fetuin only at pH 7.0 but not at 4.5, and it was higher than the activity level with sialyllactose, indicating that the N fraction was not a mixture of the S and D fractions. In Figure 2, the crude membrane fraction from the thymus (Fig. 2a) or gangliosides (Fig. 2b) was used as a natural substrate. The N fraction exhibited its highest activity at pH 5.7 (Fig. 2a). The crude membrane fraction was found to contain mainly sialoglycoproteins, as well as sialoglycolipids (gangliosides) to a lesser extent, as available substrates for sialidases. Therefore, we next examined sialidase activity with mixed gangliosides as the substrate. Because mouse gangliosides primarily contain N-glycolyl-sialic acid, and bovine gangliosides mainly contain N-acetyl-sialic acid, we prepared the total mixed gangliosides from the mouse spleen for use as a substrate (Fig. 2b). The D fraction exhibited high activity with double peaks at pH 4.1 and 5.1. In summary, the N and D fractions preferentially hydrolyzed the sialic acid bond of glycoproteins and gangliosides on the thymus-derived crude membrane, respectively, and the S fraction hydrolyzed both at a low level at neutral pH.

Table 1. Sialidase activity levels of the S, N and D fractions with various substrates
FractionSialidase activity (nmolSA released per mg protein per 2 hr)
4MU-Neu5AcFetuinSialyllactoseMucinMixed ganglio.
  1. The S, N, and D fractions were prepared from the homogenate of 20 pooled frozen thymuses from 6- to 8-week-old C57BL/6 mice as the soluble, NP40-solubilized and NP40/DOC-solubilized fractions from the crude membrane, respectively. The sialidase activities of these fractions were assayed with various substrates. Amounts of the substrate for each assay are as follows: fetuin (60 μg), sialyllactose (10 μg), mucin (50 μg), and mixed ganglio. (mixed ganglioside from bovine brain type II; 25 μg, without detergent). The released SA (sialic acid) was determined by the thiobarbituric acid method except 4MU-Neu5Ac as a substrate. Average activities for the S, N, and D fractions from three independent preparations at pH 7.0 with 4MU-Neu5Ac as a substrate were 23.2 ± 0.8, 20.4 ± 0.1, and 20.5 ± 4.9 nmoles/mg per 2 hr, respectively, and at pH 4.5 were 1.7 ± 0.1, 10.45 ± 2.05, and 26.4 ± 4.6 nmoles/mg per 2 hr, respectively.

S (soluble-cytosolic)
pH 7.024.0 ± 1.60.85 ± 0.121.74 ± 0.4800
pH 4.51.6 ± 0.200.50 ± 0.4400
N (NP40-solubilized)
pH 7.020.4 ± 0.14.42.255.480.90
pH 4.510.5 ± 2.0010.562.164.41
D (NP40/DOC-solubilized)
pH 7.017.6 ± 2.000.924.760.11
pH 4.526.5 ± 4.707.051.726.84
image

Figure 2. Sialidase activities of the S, N, and D fractions measured using natural substrates at several pH values. Conditions and enzyme fractions used for the sialidase assay were the same as those shown in Table 1. The substrate used was (a) the crude membrane fraction (4 μg sialic acid in each assay) from mouse thymus (7- to 8-week-old C57BL/6) and (b) the ganglioside mixture from pooled mouse spleen (7.5 μg sialic acid without detergent). All assays were carried out in duplicate, and the mean value was plotted. Citrate-phosphate buffer was used for pH values between 3 and 7, and PIPES buffer was used for pH values between 6 and 8.

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Candidates of the natural substrates studied with lymphoproliferation thymus

To investigate the substrates in the crude membrane, we used normal and lpr mice whose FAS gene was destroyed with a transposon [21, 22]. The thymus and lymph nodes of the lpr mice contained abnormal T cells that did not die as a result of the lack of apoptosis. Therefore, the lpr thymus was expected to have more enriched endogenous substrates. We analyzed glycoproteins on Western blots by lectin staining and gangliosides by TLC (Fig. 3). Several bands were clearly stained by SSA (Sambucus sieboldiana agglutinin, recognizes Siaα2-6 structure) in the lpr lane (indicated by arrowheads; Fig. 3a1). However, these bands were also detected by PNA (Arachis hypogaea (peanut) agglutinin, recognizes Galβ1-3GalNAc-Thr/Ser, O-glycan type; Fig. 3a2), most likely as a result of the presence of certain glycoproteins that contain multi-antennae glycans or several glycan chains on a molecule, and one has sialic acid in the terminal of the glycan, whereas the other does not. Therefore, the same molecule is detected with PNA and also with SSA lectins. More bands were also detected in lpr than in normal mice with RCA (Ricinus communis agglutinin recognizes terminal Galβ1-4 bond; Fig. 3a3), and the band marked by an arrowhead in Figure 3a4 was strongly stained after the removal of sialic acid by an acid treatment of the sheet. Thus, several candidates were demonstrated to be natural glycoprotein substrates for the sialidase. Next, we analyzed the ganglioside components of the thymus from normal and lpr mice (Fig. 3b). Bands 1 − 4 were detected more strongly in lpr than in normal mice. Next, using the crude membrane fraction from normal and lpr thymus as a substrate, we evaluated the activity level of the five types of sialidase using the plasmid-transfected COS-1 cells (Fig. 3c) to determine whether or not the lpr sample is indeed a better substrate than the normal one. All sialidases exhibited a higher level of activity against the crude membrane fraction from lpr than that from normal mice except Neu4a (L) (Fig. 3c1). The results with alternative transcripts Neu2B and Neu2C are shown in Figure 3c2. Thus, the candidate glycoproteins and gangliosides in the crude membrane described above were used as the substrates. Figure 3c3 shows the activity levels of five types of plasmid-transfected COS-1 cells with 4MU-Neu5Ac or with GM3 ganglioside. Neu2 preferred 4MU-Neu5Ac over other sialidases. When we compared the results in Figure 3c1 and c3, it is not necessarily true that the activity level with 4MU-Neu5Ac reflects the level that would be observed with a natural substrate.

image

Figure 3. Glycoproteins and gangliosides in the crude membrane fraction from normal and lpr mice. (a) Western blot analysis of the NP40-solubilized proteins from MRL/n (n) and MRL/lpr (l) thymuses. The crude membrane fraction from MRL/n (normal control) (n) and from MRL/lpr (l) thymuses were prepared and solubilized with NP40, as described in Materials and Methods, and applied to SDS-PAGE. Western blot (each lane has 4 μg protein) was stained with biotin labeled-SSA (a1), -PNA (a2), or -RCA (a3 and a4) lectin and with avidin-peroxidase. Blot a4 was acid treated to remove sialic acid before blocking the sheet. Chemiluminescence generated with enhanced chemiluminescence (ECL) plus reagent was detected using X-ray film. Size markers were included in lane M. A large arrowhead at the top indicates the position of the end of the gel. (b) Thin layer chromatography (TLC) analysis of ganglioside from MRL mice. The ganglioside mixture was extracted and purified from 500 mg thymuses of MRL/n or MRL/lpr as described in Materials and Methods. The ganglioside fraction corresponding to 3 mg protein from MRL/n (n) or MRL/lpr (l) mouse thymus was applied in each lane of TLC, separated with the C/M/0.5% CaCl2aq. (55:45:10) and reacted with resorcinol reagent. (c) Evaluation of five types of sialidase with lpr thymus crude membrane. (c1) Five types of sialidase activity using the crude membrane fraction from normal or lpr mice thymus as a substrate. The reaction mixture (25 μL) consisted of 4 mg protein of the crude membranes obtained from C57BL/6 normal (B6/n), C57BL/6 lpr (B6/l), or MRL/lpr mice (MRL/l; indicated under the horizontal axis) as the substrate for the sialidase, and 2.5 μL each of the cell homogenate of the five types of COS-1 cells transfected with the sialidase plasmid (indicated under the substrate). The mixture was incubated at pH 7.0 in 0.1 M citrate-phosphate buffer for 30 min at 37°C. The sialic acid released was measured by fluorometric high-performance liquid chromatography with malononitrile [15]. One unit of sialidase was defined as the amount of enzyme catalyzing the release of 1 nmol sialic acid/hr. Black and gray bars indicate the results of different experiments. Each bar was the average of two assays. (c2) Sialidase activity of Neu2B or Neu2C alternative transcripts with the crude membrane fraction from normal or lpr mouse thymus as a substrate. The Neu2B- or Neu2C-transfected COS-1 cell homogenates were separated into soluble and crude membrane fractions [10] and the sialidase activity of each fraction was assayed using the crude membrane fraction from normal or lpr mice thymus (4 mg protein) as the substrate, as described above. (c3) Specific activity of the cell homogenates transfected with five types of sialidase-plasmid with 4MU-Neu5Ac or ganglioside GM3. The activity of the sialidase used in (c1) was assayed with a synthetic 4MU-NeuAc substrate or ganglioside GM3 (Alexis Biochemicals, San Diego, CA, USA). The reaction mixture was incubated for 30 min at 37°C at optimal pH for each sialidase. The substrate and enzyme preparation are indicated under the horizontal axis.

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Characterization of the S, N, and D fractions by preparative IEF

The S, N, and D fractions were analyzed by IEF to characterize the sialidase in the N fraction. A representative example is shown in Figure 4. All of the sialidase-positive fractions in the S fraction exhibited activity at pH 7.0 (Fig. 4S). Thus, the S fraction contains only soluble cytosolic Neu2 sialidase that exhibits optimal activity at neutral pH. The N fraction (Fig. 4N) exhibited two components. The major fraction exhibited higher activity at pH 7.0 than at pH 4.5 and exhibited a similar or slightly higher pI than Neu2 (tubes #12 − 16), and the minor fraction exhibited higher activity at pH 4.5 than at pH 7.0 and exhibited a more acidic pI than Neu2 (tubes #6 − 7 and #10). Although the D fraction also contained two different groups, the major activity in this case was in the acidic fraction (tubes #6 − 11). After IEF fractionation, we investigated the sialidase activity using fetuin, sialyllactose, mucin, and mixed gangliosides from bovine as substrates. Fractions #9 and #10 of the S fraction, fractions #12 − 14 of the N fraction and fractions #10 and #11 of the N fraction, and #7 − 10 of the D fraction were pooled and used as S, N basic, N acidic, and D enzymes, respectively. No activity (after the removal of substrate and enzyme blanks) was detected with these substrates, although we were able to detect the activity with 4MU-Neu5As using S, N basic, N acidic, and D enzyme at 37.6, 19, 1.7, and 1.9 nmoles/mg per 2 hr at pH 7.0 and at 8.5, 3.6, 2.4, and 1.5 nmoles/mg per 2 hr at pH 4.5, respectively.

image

Figure 4. IEF analysis of the S, N, and D fractions of mouse thymus. The S, N, and D fractions were prepared from 35 pooled frozen thymuses from 4-week-old C57BL/6 mice and were analyzed by preparative IEF as described in Materials and Methods. Sialidase activity was determined using 4MU-Neu5Ac as a substrate at pH 7.0 and 4.5 with citrate-phosphate buffer. All assays were carried out in duplicate, and the mean value was plotted. The pH value of the content of each tube was plotted (—△—), and the pH values of some tubes are indicated near the activity bar.

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The pH of the same tube number of each fraction was not the same. Tube #9 of the S fraction and tube #12 of the N fraction had almost the same pH at 6.6 and 6.8, respectively. Based on the amino acid sequence of the four cloned murine sialidases [7, 3, 23, 24], the theoretical pI values were calculated as 5.67, 7.65, 6.28, and 6.06 for Neu1, Neu2, Neu3, and Neu4, respectively, by the ‘Swiss plot’ computer method based on the pKa values of amino acids [25]. If we assume that the pH values at the maximum sialidase activities separated by IEF (e.g. tube #9 in S and tubes #10 and #7 in D) correspond to the theoretical pI values, the enzyme of tube #9 in S is Neu2 (the most basic protein), and the enzymes in tubes #10 and #7 in D are Neu3/Neu4 and Neu1 (most acidic), respectively. It seems to be reasonable that tube #12 from the N fraction is Neu2. However, it is important to note that the theoretical pI may not be the actual pI of the enzymes. We conclude, however, that the basic sialidase in the N fraction that differed from Neu1, 3, and 4 is the main enzyme responsible for the membrane-associated activity toward 4MU-Neu5Ac observed in the thymus at a neutral pH.

Distribution of sialidase activity in the subcellular fractions

We next determined the distribution of sialidase activity by centrifugation under conditions used to obtain the plasma membrane. The results obtained with 4MU-Neu5Ac as a substrate at pH 7.0 and pH 4.5 are summarized in Table 2. Before sucrose fractionation, the activity level at pH 7.0 was higher than that of pH 4.5. After sucrose fractionation, the activity at pH 7.0 was lower than that at pH 4.5 in all of the fractions, and the recovery of the activity at pH 7.0 and that of the proteins was low. This result may be explained as follows: a portion of the activity at pH 7.0 with 4MU-Neu5Ac in the crude membrane fraction might result from the interaction between Neu2 and the membrane structure, and Neu2 was dissociated from the membrane in 35% sucrose or the washing process. Conversely, no loss in activity was observed at pH 4.5 because the membrane-bound enzymes exhibited the activity.

Table 2. Results obtained with 4MU-Neu5Nc as a substrate at pH 7.0 and pH 4.5 and sialidase activity level of each fraction and recovery of activity and protein
 Sialidase activityRecovery of activity (%)Recovery of protein (%; average of two cases)
pH 7.0 / pH 4.5pH 7.0 / pH 4.5
  1. The cell homogenate of the thymus was prepared as previously reported [4], centrifuged at 450g for 10 min and the precipitate (450P) and supernatant (450S) were separated. The 450S was centrifuged at 105,000g for 1 hr, and S (105k sup) and CM (the crude membrane fraction, 105k ppt) were obtained. The crude membrane fraction was suspended in PBS by sonication and overlaid on 35% sucrose. The mixture was separated by centrifugation at 24,000g for 1 hr with a Hitachi swing rotor SW28 into upper, middle, lower and precipitate fractions.

  2. The sialidase activity of each fraction was assayed with 4MU-Neu5Ac as a substrate at pH 7.0 and 4.5 (nmoles of 4MU released/mg per 2 hr). Recovery of the activity and the protein were calculated to the total homogenate as 100%.

  3. 105k, 105,000g; ppt, precipitate; sup, supernatant.

Total homog.14.9 / 10.4100 / 100100
450P2.3 / 3.03.9 / 5.915.7 ± 2.8
450S14.3 / 14.588.5 / 10578.2 ± 10.1
S (105k sup)10.8 / 3.851.4 / 21.247.3 ± 5.1
CM (105k ppt)20.6 / 18.746.5 / 60.433.7
Upper4.8 / 11.21.3 / 3.72.5 ± 0.6
Middle21.5 / 63.45.6 / 19.52.7 ± 0.1
Lower21.5 / 73.81.9 / 7.51.2 ± 0.2
Ppt10.8 / 27.89.8 / 29.612.7 ± 2.7

Sialidase activity was also analyzed in the middle layer fraction that was expected to enrich with plasma membrane (Table 3). Among the substrates tested, only fetuin exhibited a higher value at pH 7.0 than that at pH 4.5. Exp. #1 in Table 3 has a higher ratio of alkaline/acid phosphatase activity and a higher level of sialidase activity with fetuin at pH 7.0 than Exp. #2. This result might suggest that the enzyme that released sialic acid from fetuin at pH 7.0 existed in the plasma membrane.

Table 3. Sialidase activity level in the middle (plasma membrane-rich) fraction with different substrates
 Sialidase activity with ‘middle’ fraction (nmol/mg per 2 hr)
Exp. #1Exp. #2
pH 7.0 / pH 4.5pH 7.0 / pH 4.5
  1. See footnote 1 of Table 2.

  2. Released sialic acid was determined by the thiobarbituric acid method.

  3. Exp. #1 was the result obtained in the conditions described in Table 2. Exp. #2 was the alternative result obtained by overlaying the precipitate of 34,000g for 15 min on 35% sucrose (in this case, some of the plasma membrane-rich fraction was missing from the precipitate).

  4. Alk./Acid P, ratio of alkaline phosphatase activity/acid phosphatase activity (alkaline or acid phosphatase is a marker enzyme of plasma membrane or lysosome, respectively); ND, not done.

4MU-Neu5Ac11.8 / 38.88.6 / 55.1
Fetuin6.6 / 03 / 1.2
Sialyllactose3.6/22.61 / 54
GM3ND / ND20.5 / 126.7
Oroso mucoidND / ND0 / 0
Alk. / Acid P88.518.8

Main membranous sialidase at neutral pH containing Neu2 is associated with the membrane structure but is not anchored

As mentioned above, Neu2 appeared to interact with the membrane in the crude membrane fraction. We have already reported that the crude membrane fraction from COS-7 cells that were transfected with the Neu2-plasmid released its sialidase activity following three washes with 10 mM Tris-HCl, pH 7.0 [10]. Thus, we carried out the same experiment with the crude membrane fraction from the mouse thymus. The results were similar to those observed with the COS cells; the first wash had little or no effect on the activity, whereas three washes reduced the activity to the residual level (approximately 40%) of the crude membrane fraction after solubilization with NP-40 (Fig. 5a). We also detected sialidase activity in the washed solution (data not shown). These observations suggest that sialidase solubilized with NP40 is associated with the membrane structure of the thymus but not anchored in the membrane by a signal peptide or GPI-anchor, for example, trans-sialidase [26].

image

Figure 5. Characteristics of the major sialidase in the crude membrane fraction. (a) Washing the crude membrane. The crude membrane fraction from two C57BL/6 mice (7 weeks old), obtained by centrifuging the homogenate at 90,000g, was suspended in 10 mM Tris buffer, pH 7.0, and an aliquot (M1) was assayed for sialidase activity with 4MU-Neu5Ac. The remainder was re-centrifuged at 90,000g for 30 min to obtain the second precipitate (M2), and the same procedure was repeated to obtain the M3 − M5 fractions. Each precipitate was resuspended in the same volume of 10 mM Tris-HCl, pH 7.0, before each centrifugation. Activity of each suspension was assayed with the same volume of the aliquot M1. Relative activities (%) to M1 were plotted (—●—). As a control, the crude membrane fraction (M1) was solubilized with NP-40 solubilizing buffer for 30 min, and after centrifuging at 90,000g for 30 min, the activity of the residual precipitate was assayed (—▪—). (b) Immunoprecipitation of the S and N fractions with anti-Neu2 antibody. Each of the S and N fractions (25 µL) was incubated for 1 hr on ice with 1.5 µL anti-Neu2 rabbit polyclonal antibody (black bar) or non-immunized rabbit serum as the negative control (gray bar) in a final volume of 30 μL. The incubated mixture was added to protein A-sepharose beads (50 μL of a 20%, v/v suspension) that were packed in a small conical tube by centrifugation, and the mixture was mixed for 2 − 5 hr in a rotator at 10°C. The tube was centrifuged, and the supernatant was assayed for sialidase activity using 4MU-Neu5Ac in citrate-phosphate buffer, pH 7.0, as a substrate. With the commercial anti-Neu2 goat polyclonal antibody, protein-G sepharose beads were used instead of protein A-sepharose beads.

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To determine whether Neu2 is responsible for the sialidase activity in the crude membrane fraction at neutral pH with 4MU-Neu5Ac, we carried out an immunoprecipitation experiment. Incubation of anti-Neu2 rabbit polyclonal antibody with the S or N fraction reduced a portion of the activity (12% or 7% reduction, respectively; Fig. 5b), although the extent of the reduction with the S fraction (positive control) was not sufficient. We examined the same experiment with a commercially available anti-Neu2 goat polyclonal antibody purchased from Santa Cruz Biotechnology, but the reduction of the activity was less than that in the above experiment. At this time, we cannot say definitively that Neu2 is responsible for the sialidase activity in the crude membrane fraction at neutral pH with 4MU-Neu5Ac, although we can state that Neu2 is partially responsible for this activity.

SMXA recombinant mouse studies suggest that the Neu2 gene controls sialidase activity in the crude membrane fraction at pH 7.0

Next, we tried to access how the sialidase gene controls the activity in the crude membrane fraction at pH 7.0 using a recombinant inbred strain of SM and A mice (SMXA). We reported that the level of sialidase activity in the soluble fractions from 22 substrains of SMXA mice exhibited good correlation with the D1Mit8/9 marker, which is located close to the Neu2 gene on mouse chromosome 1 [5]. Therefore, in the present study, we determined the activity level of the crude membrane fraction from the 22 substrains at pH 7.0 and observed which gene best correlated with the activity. Although the activity levels in the crude membrane fractions from the parental SM and A/J mice did not exhibit any differences at neutral pH, the membrane-bound activities from 22 substrains of the SMXA were well correlated with the genotype of the D1Mit8/9 locus, except for one substrain (SMXA #19; Fig. 6). In contrast, the activities did not correlate with the genotype of Neu1, Neu3, or Neu4. This finding shows that the sialidase activity of the crude membrane fraction at pH 7.0 is most likely regulated by the Neu2 gene by an unknown mechanism.

image

Figure 6. Level of sialidase activity in thymus crude membrane at pH 7.0 from SMXA recombinant mice. Sialidase activities were assayed using 4MU-NeuAc at pH 7.0 with the crude membrane fraction of the thymuses from SMXA recombinant mice. Values are the average of two replicates. The mice were 9 − 11 weeks old. A and S under the strain number indicate the genotype of each strain at the marker of D1Mit 8/9 on chromosome 1 (near the Neu2 gene), at the marker of the Neu1 locus on chromosome 17, at the marker of Hbb on chromosome 7 (near the Neu3 gene) and at the marker of D1Mit 10 or D1Mit 11 on chromosome 1 (near the Neu4 gene). Underlined A and S indicate the un-accord genotype with the S or A group at D1Mit8/9 on chromosome 1, if we assume that the S group has a lower activity level and the A group has a higher activity level. White and gray bars indicate the activity level of the S and A groups of the SMXA recombinant mouse at D1Mit8/9 on chromosome 1, respectively, and black and marble bars indicate the activity level of the parental A/J and SM/J mice, respectively.

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Sialidase activity in the sorted cells

Finally, we attempted to identify the sialidase-positive cells in the thymus. First, we separated the cells from the thymus into T cells and non-T cells. Next, T cells were separated into PNA-aggregated and PNA-unaggregated T cells. The sialidase activity at pH 7.0 and pH 4.5 in each of the cell types was investigated using 4M-Neu5Ac as a substrate (Table 4). Non-T cells (‘neu-medullocytes’ (5) are most likely contained in this fraction) exhibited clearly higher activity than T cells at pH 7.0 and 4.5 (Table 4). Next, T cells were sorted into DP cells (corresponding to the PNA-aggregated cells in Table 4), CD4+ SP, CD8+ SP, and DN cells (these three cell types correspond to the PNA-unaggregated cells in Table 4) using a cell sorter, and the activity level of sialidase was determined (Table 5). Few differences in activity were observed in the soluble fractions among the four types of T cells, whereas the crude membrane fractions from CD8+ SP cells exhibited slightly higher activity than the other T cell types.

Table 4. Sialidase activities in PNA-aggregated T cells, PNA-unaggregated T cells and non-T cells
 Sialidase activity (4MU released nmol/mg protein per 2 hr)
Activity at pH 7.0Activity at pH 4.5
Soluble fr.Membrane fr.Soluble fr.Membrane fr.
  1. The cell suspension from two fresh thymuses of A/J (7-week-old) was passed through three layers of nylon mesh, and T cells (passed cells) and non-T cells (on the mesh) were obtained. The T cells (2 × 108/mL) were incubated with an equal volume of PNA solution (1 mg/mL) for 10 min. PNA-aggregated T cells (PNA-agg. T) were separated from PNA-unaggregated single cells (PNA-unagg. T) by overlaying the cells on 20% FCS solution [5]. PNA-unaggregated cells remained on the top of the solution. PNA-agg. T and PNA-unagg. T cells were washed with 0.1 M lactose/RPMI medium and then with PBS. These cells were lysed by freezing and thawing three times and sonicated in 0.1 mM CaCl2 containing 200 μg PMSF/mL for 15 s two times at the lowest power condition and centrifuged at 18,000g for 30 min. The soluble and precipitate membrane fractions were then assayed for sialidase activity with 4MU-Neu5Ac.

PNA-agg. T1.78 ± 0.001.64 ± 0.300.82 ± 0.411.28 ± 0.18
PNA-unagg. T0.78 ± 0.401.86 ± 0.210.78 ± 0.261.04 ± 0.76
Non-T9.16 ± 2.105.23 ± 0.520.60 ± 0.478.79 ± 0.34
Table 5. Relative sialidase activity of the soluble and crude membrane fractions of the sorted T cells using 4MU-Neu5Ac as a substrate
 Relative activity (%) to the activity with CD4
Exp. #1Exp. #2
Soluble fr.Membrane fr.Soluble fr.Membrane fr.
  1. T cells were sorted into DP, DN, CD4+ SP, and CD8+ SP cells using a cell sorter, and soluble and membrane fractions were prepared as described in Table 4. The sialidase activity was assayed at pH 6.6 with citrate-phosphate buffer and shown as the relative activity to that of CD4+ SP. Exp. #1 and #2 were the results obtained with 2.5 and 5 × 105 cells, respectively. The DN fraction of Exp. #2 was contaminated with some red blood cells. Relative activities of the crude membrane fraction compared to the soluble fraction with CD4+ SP cells were 92% and 98% in Exp. #1 and #2, respectively.

DN1579184167
DP12312492.4112
CD8129250112158
CD4100100100100

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The present study aimed to determine whether soluble sialidase Neu2 is responsible for the high level of sialidase activity at neutral pH with 4MU-Neu5Ac in the crude membrane of the thymus. We examined this question from several perspectives and determined that it is likely that the high level of membranous sialidase activity at neutral pH is due to soluble sialidase Neu2 associated with the cellular membranous fraction by some unknown mechanism. This conclusion is supported by the results obtained by IEF analysis (Fig. 4), the membrane fraction washing experiment (Fig. 5a), and the activity-regulating gene results suggested by our experiments with SMXA recombinant mice (Fig. 6). Unfortunately, we could not show a clear-cut result with the anti-Neu2 antibody used in our immunoprecipitation experiments or by Western blot analyses. One point to be stressed is that when using 4MU-Neu5Ac as a substrate for the assay of sialidase activity, the results will primarily reflect Neu2 activity as shown in Figure 3c. Another important point is that we found that the plasma membrane-rich fraction from the mouse thymus contained a sialidase that removed sialic acid from fetuin at pH 7.0 but not at pH 4.5. This activity is unique among the sialidases to date, indicating that this enzyme perhaps removes sialic acid from a glycoprotein on another T cell or the glycoprotein existing near the sialidase on the plasma membrane. This sialidase must have an important physiological function.

Next, we discuss the physiological function of the cytosolic sialidase in the thymus. In terms of catabolism, a cytoplasmic peptide: N-glycanase (PNGase) is reported to release glycans from newly synthesized misfolded glycoproteins [27]. The released glycans are hydrolyzed by cytosolic glycosidases and, very recently, the dual functions of cytosolic glycosidase during glycan catabolism and apoptotic signaling were reported [28]. Although the function of glycosidase as an apoptotic signal was reported [29, 30], this function is unclear in the thymus. In the thymus, apoptosis occurs frequently and the degradation of glycoproteins by cytosolic glycosidases must play an important role. Thus, it might be important that Neu2B sialidase in the thymus has a higher activity with sialyllactose compared to Neu2C sialidase in the muscle (5.8-fold for α2-6sialyllactose and 8.6-fold for α2-3sialyllactose [10]). Another point that we wish to stress is that a transit state of some cellular proteins may carry out some important physiological function. For example, it may be possible that the association of Neu2 with the membrane structure or Neu2's stability is affected by some conditions; that is, it is metabolically stable under some conditions but becomes unstable upon a change in the cell's state, for example, apoptosis. Biochemically, the labile character of the murine sialidase (e.g. 1.1 − 1.3 hr of half-life) was reported [31]. The excess six amino acids (if they are translated in the enzyme) might affect cytosolic Neu2 during this type of physiological change. For example, it associates with the membrane structure and then dissociates after carrying out a specific function, as we discussed in the case of tetraspanin [16]. Indeed, we demonstrated the expression of active Neu2 on the plasma membrane by X-NANA active staining of Neu2B transfected COS cells [10]. The sialidase activity with fetuin in the plasma membrane may be one such example. The majority of lysosomal sialidase Neu1 relocalizes from the lysosomes to the cell surface during the differentiation of monocytes [32]. Studies of type B Neu2 in the thymus may provide additional information.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

More than half of this study was carried out at the Institute of Immunology/Genetic Medicine, Hokkaido University, with the support of a Grant-in-Aid for Scientific Research (C) [2] (07808069) from the Ministry of Education of Japan (1995 − 1996) until 2001, when S.K.-O, N.D., and M.F. were present. The rest of the study was conducted at the laboratory of Prof. T. Koda (Hokkaido University). Some of the study was conducted at Tohoku Pharmaceutical University (Sendai). We are grateful to Prof. J. Inokuchi (Tohoku Pharmaceutical University, Sendai) for introducing Dr K. Kabayama and Dr S. Go to conduct TLC and Western blot analyses, respectively. We are grateful to Ms H. Matsukawa (deceased) for her work on Figure 6 when she was at the Institute of Immunology, Hokkaido University. We are grateful to Dr K Azumi (homogenizer), Prof. Y. Igarashi, and Dr S. Mitsutake (ultracentrifuge, sonicator), and Prof. S. Nishimura (spectrofluorometer) at Hokkaido University for the use of their laboratory equipment, as well as the Central Institute of Isotope Science for storing materials at –70°C after S.K.-O.'s retirement. We are grateful to Prof. M. Nishimura (retired from Nagoya University) for his kind donation of SMXA recombinant mice and Dr K. Ogasahara (Institute for Protein Research, Osaka University) for suggesting the use of the Swiss Plot method to calculate the pI values.

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

No authors have any conflict of interest to disclose.

REFERENCES

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  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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