Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis

Akira Seko, Ken Kitajima1, Takashi Iwamatsu2, Yasuo Inoue3,4 and Sadako Inoue3

Department of Biochemistry, Sasaki Institute, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan, 1Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan, 2Department of Biology, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-0001, Japan and 3Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan, R.O.C.

Received on December 2, 1998; revised on February 26, 1999; accepted on March 1, 1999

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish (Oryzias latipes). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the Km for L-hyosophorin was 44 µM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of theL-hyosophorin.

Key words: de-N-glycosylation/peptide:N-glycanase (PNGase)/ fish embryo/diversity in PNGase

Introduction

Accumulation of free N-linked complex-type sialooligosaccharides was first reported in the cytosol of unfertilized eggs of Plecoglossus altivelis, a fresh water trout (Ishii et al., 1989). The free glycan chains were bi-, tri-, and tetra-antennary structures that had di-N-acetylchitobiosyl units at the reducing termini. Subsequently, similar free oligosaccharides were found to occur widely in unfertilized eggs and embryos of fish such as Tribolodon hakonensis (a dace) (Inoue et al., 1989), Oryzias latipes (medaka fish) (Iwasaki et al., 1992), and Scyliorhinus caniculus (Plancke et al., 1996), and in hen eggs (Seko et al., 1997). Following these discoveries, it became evident that phosvitins, polyphosphorylated glycoproteins in the yolk of oviparous eggs were the most likely precursor glycoproteins of the free glycan chains because their sugar chains were uniquely species-specific and shared structural features in common with the free oligosaccharides that accumulated in each species of P.altivelis, T.hakonensis, and O.latipes (Iwasaki et al., 1992).

The accumulation of free penta-antennary N-glycans containing the di-N-acetylchitobiosyl structure at their reducing termini in the embryos of Paralichthys olivaceus (flounder) and O.latipes was also reported (Seko et al., 1989, 1991a). The structure of these glycan chains was typical of those found in cortical alveolar glycoproteins (hyosophorin) of fish eggs but not found in phosvitins (Seko et al., 1989; Taguchi et al., 1993; Inoue and Inoue, 1997). The glycan chains characteristic of hyosophorin were not detected in their free form in unfertilized eggs, and we showed that the free glycan chains accumulated in embryos of O.latipes between fertilization and early gastrula (Seko et al., 1991a). Although the significance of the release and accumulation of free glycans during vitellogenesis and embryogenesis remains unclear, the observed phenomena raised the possibility that de-N-glycosylation of certain glycoproteins may be an important posttranslational remodification process of proteins (Inoue, 1990; Iwasaki et al., 1992; Suzuki et al., 1994b).

The presence of di-N-acetylchitobiosyl structure at the reducing termini of accumulated free glycan chains strongly suggested the involvement of peptide:N-glycanase (EC 3.5.1.52, PNGase) in de-N-glycosylation of fish egg and embryo glycoproteins. Indeed, we showed the first occurrence of animal PNGase in the embryos of O.latipes (Seko et al., 1991b). More recently we have shown ubiquitous occurrence of PNGase activities in mammalian cultured cells, mouse organs, and hen oviduct (Suzuki et al., 1993, 1994, 1997a; Kitajima et al., 1995). Identification of endogenous substrates for the PNGase activities found in specific cells and organs is an important goal in understanding the biological function of PNGase. To this end, we have determined that overglycosylated ovalbumin is an endogenous substrate for the PNGase activity in hen oviduct, and have hypothesized that this de-N-glycosylation is of physiological significance (Suzuki et al., 1997). More recently, we have shown further diversity in the PNGases by identifying a second neutral PNGase activity that is expressed in L-929 cultured cells during the log phase of growth (Chang et al., 1997). The enzymatic properties of this enzyme were discovered to be distinctly different from those previously described for the PNGase purified from confluent L-929 cells (Suzuki et al., 1995).

To seek further understanding about the developmental expression and physiological significance of PNGase, we have examined in this study the expression of PNGase activity at different stages of development during fish embryogenesis. These studies have revealed that PNGase expression coincided both spatially and temporally with the level of cell-specific glycoproteins that are physiological substrates for PNGases, and that free glycan chains are formed by the action of these enzymes. Further, we have identified two discrete types of PNGases in developing embryos of medaka fish, O.latipes, which we have designated as acid and neutral PNGase M, respectively. The two activities differed in two major ways. First, the optimum pH where the enzymes were most active differed, with the acid PNGase showing an optimum pH between 3.5-4.0, while the neutral enzyme was most active between pH 7.5-8.5. Second, the acid PNGase was maximally expressed at developmental stage 18, whereas the neutral enzyme activity was only detected in the blastoderms obtainable from the embryos at stage 11. Based on colocalization studies, we propose that acid PNGase M is involved in catabolism of glycosylated phosvitins, whereas neutral PNGase M functions in the modification of cortical alveolar glycoproteins during early embryogenesis (cf. Inoue and Inoue, 1997).

Results

Two distinct PNGase activities are expressed at different stages of development during embryogenesis in Oryzias latipes

Our previous studies showed that PNGase activity was expressed in the embryos, which are comprised of a mixture of those at stages 10 to 28, of O.latipes (medaka fish) as the first example of the occurrence of PNGase in animal cells (Seko et al., 1991b). This enzyme activity was suggested to be responsible for the de-N-glycosylation of L-hyosophorin during early development between fertilization and early gastrula (Seko et al., 1991a). However, when whole embryos and the isolated blastoderms were assayed for the PNGase activity using [14C]fetuin glyco-peptide II as substrate, two distinct types of PNGase activity were detected as shown in Table I. These activities showed clear difference in their localization, optimum pH, and developmental expression at different stages of embryogenesis.

Table I. PNGase activities in the whole embryos and the isolated blastoderms at the middle blastula (stage 11) and the formation of optic buds (stage 18)
  Middle blastula (stage 11) Formation of optic buds
(stage 18)
pH 4.0 pH 7.5 pH 4.0 pH 7.5
  (pmol/min/100 embryos) (pmol/min/100 embryos)
Whole embryos 0.0 0.0 0.38 0.0
Blastodermsa 0.0 0.42    
The [14C]fetuin glycopeptide II (0.55 nmol) was incubated with homogenates of whole embryos or bladstoderms at 25°C for 24 h, and the product peptide was quantitated.aDue to the technical difficulties inherent in isolating blastoderms from embryos at stages later than 11, the present study only allowed the detection of neutral PNGase activity in the isolated blastoderms at earlier stages (cf. Figure 4).

The blastoderms isolated from the middle blastula embryos (stage 11) contained the PNGase activity which was active at pH 7.5, but not at pH 4.0. In contrast, whole embryos did not express any detectable PNGase activity at stage 11, and only showed activity at the formation of optic buds (stage 18) at pH 4.0. The PNGase activity found in the blastoderms was not detectable in the homogenate of the whole embryos, possibly because the activity at neutral pH was inhibited by a large body of yolk glycoproteins in the whole embryos. Therefore, as shown below, the PNGase activity that had been identified in the previous study in the whole embryos turned out to be the enzyme with acidic pH optimum. The enzymes with acidic and neutral pH optimum were designated as acid PNGase M and neutral PNGase M, respectively.

Identification and characterization of neutral and acid PNGase M activities

Identification of neutral PNGase activity in blastoderms

The supernatant fraction prepared from the blastoderms derived from 3000 embryos at blastula stage was incubated at pH 7.5 with 14C-labeled asialofetuin glycopeptide II as a substrate at 25°C for 24 h. The reaction mixture was then subjected to Sephadex G-50 gel filtration (Figure 1). A peak of hexose-containing material (fraction I) and that of radioactive material (fraction II) were separated from the intact glycopeptide that was eluted in fractions 53-58.


Figure 1. Gel filtration on a Sephadex G-50 column of the hydrolysate of fetuin asialoglycopeptide II by the enzyme fraction obtained from the blastoderms of O.latipes at pH 7.5. One-ml fractions were collected and monitored for radioactivity (solid circles) and the phenol-sulfuric acid reaction, A490(open circles). Void volume corresponds to fraction 26. Black arrow indicates the elution position of intact fetuin asialoglycopeptide II.

No hexose-containing material appeared in fractions 50-70 when the control reaction mixture without the 14C-labeled substrate was incubated and subjected to Sephadex G-50 gel filtration. This indicated that the pooled fraction I (Figure 1) was not derived from endogenous glycoconjugates, but rather from the added [14C]fetuin glycopeptide substrate. The radiolabeled fraction II (Figure 1) was isolated and chromatographed on a Sephadex G-25 column, where 77% of the radioactivity eluted at the void volume (Vo) and the residual 23% eluted at more retarded position. The Vo [14C]-labeled product was analyzed by thin-layer chromatography (TLC) and paper electrophoresis, as shown in Figure 2. On both analyses, the radiolabeled product migrated with the same mobility as the authentic [14C]pentapeptide, [14C]Me2Leu-Ala-Asp-CmCys-Ser. The 23% of radioactivity that was retarded on the Sephadex G-25 column was identified as [14C]Me2Leu, by paper chromatography (result not shown). The fraction I (Figure 1) was analyzed for its sugar composition (Table II). Before NaBH4 treatment, the sugar composition of fraction I was identical with that of the intact glycopeptide, whereas after NaBH4 treatment, one GlcNAc residue was lost concomitant with the appearance of one GlcNAc-ol residue. These results show that the glycan chain in fraction I was a free oligosaccharide chain having di-N-acetylchitobiosyl structure at the reducing terminus, and confirm the presence of a PNGase activity in O.latipes blastoderms at the mid-blastula (stage 11).


Figure 2. Thin layer chromatographic identification of the radiolabeled products resulting from digestion of fetuin [14C]asialoglycopeptide II with the PNGase fraction prepared from the blastoderms of O.latipes. (a) TLC mobility of the 14C-labeled reaction product. Each sample was spotted on a Kiesel gel 60 silica gel plate, and developed in pyridine/ethylacetate/acetic acid/water (5/5/1/3, v/v/v/v) for 2.5 h. Radioactivity was visualized on a Bio-Imaging analyzer. (b) Detection of the [14C]reaction product on paper electrophoresis. Each sample was spotted on a Whatman 3 MM paper, electrophoresed in pyridine/acetic acid/ water (5/0.2/95, v/v/v; pH 6.5) at 13 V/cm for 2.5 h, and visualized by a Bio-Imaging analyzer. Lane 1,fetuin [14C]asialoglycopeptide II; lane 2, authentic [14C]pentapeptide;lane 3, fraction II after Sephadex G-25 gel filtration; lane 4, [14C]pentapeptide-GlcNAc; lane 5, [14C]Me2Leu.

Table II. Carbohydrate compositions of fetuin asialoglycopeptide II and fraction I obtained from Sephadex G-50 column chromatography after digestion of 14C-fetuin asialoglycoprotein with PNGase from blastoderms (stage 11) at pH 7.5
  Fetuin asialoglycopeptide II Fraction I
Before After
Man 3.0 3.0 3.0
Gal 2.9 2.9 2.7
GlcNAc 5.0 5.1 4.2
GlcNAcol n.d.a n.d.a 0.95
The analyses were carried out before and after reduction with NaBH4. Molar ratios of monosaccharides are expressed relative to mannose set equal to 3.0.
aNot detected.

The neutral PNGase in blastoderms can act on L-hyosophorin

To determine if [14C]L-hyosophorin ([14C]Me2Asp-Ala-Ala-Ser-Asn(CHO)-Gln-Thr-Val-Ser) was similarly hydrolyzed by PNGase in the supernatant fraction prepared from stage 11 blastoderms, the 14C-labeled product formed after incubation was analyzed by TLC and paper electrophoresis, as described above. These results showed that the 14C-labeled product migrated identically with the authentic [14C]nonapeptide standard on both TLC and paper electrophoresis (results not shown). On the basis of these results, we conclude that the PNGase activity in stage 11 blastoderms can catalyze the release of N-linked oligosaccharide chains from L-hyosophorin.

Effect of pH on the enzymatic properties of the PNGase, neutral PNGase M from blastoderm at stage 11

The effect of pH on the PNGase activity derived from stage 11 blastoderms is shown in Figure 3a. The maximum activity was obtained at pH 7.5-8.5 and essentially no activity was detected below pH 5 or above pH 10. Because of this neutral pH optimum, we designated this enzyme as neutral PNGase M. Release of the [14C]peptide increased linearly up to 4 h at 25°C at pH 7.5.


Figure 3. Effect of pH on (a) neutral and (b) acid PNGase M activities obtained from O.latipes blastoderms at stage 11 (neutral enzyme) and whole embryos at stage 18. (a) Buffers used were sodium citrate (pH 3.70), sodium acetate (5.01-6.50), HEPES-NaOH (6.84-8.16), Tris-HCl (8.00-9.00), and glycine-NaOH (9.27-10.30). (b) Buffer used were sodium citrate (pH 2.83-5.00) and sodium acetate (5.01-6.50).


Figure 4. Neutral PNGase M (solid circles) and acid PNGase M (open circles) activities in the embryos of O.latipes at various developmental stages. Stage numbers correspond to 8 cells (stage 5), 32 cells (stage 7), late morula(stage 9), middle blastula (stage 11), early gastrula (stage 13), formationof optic buds (stage 18), circulation in vitelline veins (stage 25), and black pigmentation of eyes (stage 28).

Effect of reaction products and thiol reagents on the acid and neutral PNGase M activities

Assays to determine the effect of the PNGase M catalyzed reaction products and the thiol reagents, monoiodoacetic acid (MIA), p-chloromercuribenzoic acid (PCMB) and phenylmethyl-sulfonyl fluoride (PMSF), were carried out to assay if they inhibited the acid and neutral PNGases M activities. The results of these studies provided the following further evidence for distinct differences between the two forms of the enzymes. First, the neutral PNGase M activity was not inhibited by either the reaction products, peptide-free N-linked glycan or the de-N-glyco-sylated peptide at 1 mM. In contrast, the acid PNGase M activity was inhibited 92% by 1 mM L-hyosophorin-derived nonapeptide and only 5% by the L-hyosophorin-derived free glycan. A second difference was revealed by the effect of the SH group-modifying reagents, MIA and PCMB, on activity. The neutral PNGase M activity, for example, was inhibited 89% and 82% by 5 mM MIA and 0.5 mM PCMB, respectively, but was not inhibited by 5 mM PMSF. The acid PNGase M activity, in contrast, was essentially unaffected by any of these thiol reagents when tested at the same concentrations. The activity of the acid PNGase M was nearly completely inhibited by 5 mM FeCl3 and 5 mM CuSO4, while 5 mM CaCl2, MgCl2, MnCl2, or ZnCl2 had no effect on activity. The effect of these divalent cations on the neutral PNGase M activity showed that the presence of 5 mM FeCl3, CuSO4, or ZnCl2 caused substantial inhibitory effect and only 10-20% residual activities were observed whereas CaCl2, MgCl2, and MnCl2 had almost no inhibitory effect.

Developmental change of acid and neutral PNGase M activities

The developmental expression of the activity in the blastoderms from the 32 cells to the early gastrula stage was investigated. Unfortunately, the activities at later stages could not be determined, because it was too difficult to surgically isolate blastoderms after the early gastrula stage. At this stage, the blastoderm expands toward the vegetal pole and surrounds most of the yolk sphere (epiboly). Neutral PNGase M activity was found in the blastoderms at the every stage examined (Figure 4). The enzyme activity at stage 11 was 4.2 × 10-3 pmol/min/embryo when using [14C]fetuin glycopeptide II as a substrate. In contrast, the acid PNGase M activity was not detected between the 8 cell (stage 5) and late gastrula stage, but rather showed a sharp increase between stages 18-28 (Figure 4). In the ovaries of O.latipes, PNGase activity having an acidic pH optimum was detected at a level corresponding to 28% (on wet weight basis) of the activity in stage 28 (results not shown). This appears to be the same enzyme as the acid PNGase M partially purified from the embryos, as described previously (Seko et al., 1991b).

Substrate specificity

The relative rates of hydrolysis of various glycopeptides and glycoproteins catalyzed by acid PNGase M was summarized in Tables III and IV, respectively. Glycopeptides containing a complex, hybrid, or high-mannose type N-linked glycan chain were good substrates, whereas Asn-oligosaccharide, GlcNAc-peptide, and the stem bromelain glycopeptide which possesses a-fucose residue linked to the C-3 of the proximal GlcNAc (Ishihara et al., 1979), were not hydrolyzed. On the other hand, the Bence-Jones glycoprotein (Wh [lambda])-derived glycopeptide, which possesses [alpha]-fucose residue linked to the C-6 of the proximal GlcNAc (Ohkura et al., 1985), was hydrolyzed. This strict specificity for fucose residues is similar to that of PNGase F (Tretter et al., 1991). As for glycoproteins, acid PNGase M was able to release glycan chains from O.latipes H-hyosophorin and glycophosphoproteins (MU-1 and MU-2), but not from fetuin or ovalbumin.

Table III. Relative rate of hydrolysis of various glycopeptides catalyzed by acid PNGase M
Substrate Glycan structure Relative rate (%)
O.latipes L-hyosophorin Complex-type pentaantennary
(Fuc2.1Man3.0Gal15.8GlcNAc9.6NeuAc4.7)
100
Ovalbumin glycopeptide (hybrid) Hybrid-type (Man4.0Gal0.5GlcNAc5.6) 166
Ovalbumin glycopeptide (high Man) High mannose-type (Man5.5GlcNAc2.0) 152
Ovalbumin GlcNAc-peptide GlcNAc 0
Glycoasparagine (ovalbumin GP-IIIA) Hybrid-type (Man5.0GlcNAc4.0) 0
Bence-Jones Wh [lambda] glycopeptide [alpha]1->6-fucosylated complex-type biantennary
(Fuc0.85Man3.0Gal2.0GlcNAc4.9NeuAc1.7)a
181
Fetuin glycopeptide II Complex-type triantennary
(Man3.0Gal3.0GlcNAc5.0NeuAc3.0)
161
Stem bromelain glycopeptide [alpha]1->3fucosylated Xyl-containing
(Xyl1.0Fuc0.86Man2.6GlcNAc2.0)b
0
Defucosylated stem bromelain glycopeptide Xyl-containing (Xyl0.52Man2.6GlcNAc2.0)c 50
aThe glycan has the bisecting GlcNAc residue and an [alpha]1->6-linked Fuc on the proximal GlcNAc.
bThe glycan has the [alpha]1->3-linked Fuc residue attached to the proximal GlcNAc.
cThe [alpha]1->3-linked Fuc was removed by mild acid hydrolysis.

Table IV. Relative rate of hydrolysis of various glycoproteins catalyzed by acid PNGase M
Substratea Glycan structure Molecular weight
(K)
Relative rate
(%)
O.latipes H-hyosophorin Complex-type pentaantennary 128 100
Fetuin Complex-type bi- and triantennary 48.4 0
Ovalbumin Hybrid- and high mannose-type 45 0
O.latipes GPP MU-1 Complex-type bi- and triantennary 25 199
O.latipes GPP MU-2 Complex-type bi- and triantennary 10 176
aGPP, Glycophosphoprotein.

Inhibition of the acid PNGase-catalyzed de-N-glycosylation of [14C]L-hyosophorin by the de-N-glycosylated peptide product

We measured the initial reaction rates for the acid PNGase M-catalyzed hydrolysis of [14C]L-hyosophorin at various concentrations of [14C]L-hyosophorin as substrate. We estimated the Michaelis constant (Km) as 44 µM by a Lineweaver-Burk plot (Figure 5; corresponding to the line that [14C]penta-peptide was at 0 µM (solid circles)). The Vmax value was 12.1 nmol/min/mg of protein. The effect of fetuin glycopeptide II-derived, de-N-glyco-sylated pentapeptide (Leu-Ala-Asp-CmCys-Ser), which was produced by PNGase digestion, was also investigated (Figure 5). In the presence of the pentapeptide at 20, 40, or 60 µM, the enzymatic reaction was competitively inhibited, and the apparent Km changed to 110, 180, or 250 µM, respectively. From these data, the inhibitory constant (KI) of the pentapeptide was calculated to be 13 µM. L-hyosophorin-derived, de-N-glycosylated nonapeptide (Asp-Ala-Ala-Ser-Asp-Gln-Thr-Val-Ser) also competitively inhibited acid PNGase M activity and the KI was calculated to be 110 µM.


Figure 5. Lineweaver-Burk plots for acid PNGase M-catalyzed de-N-glycosylation of [14C]L-hyosophorin in the absence and presence of fetuin glycopeptide II-derived pentapeptide (Leu-Ala-Asp-CmCys-Ser). vi, initial reaction rate (nmol/min/mg of protein); [S], concentration of [14C]L-hyosophorin (µM). Initial reaction rate was determined in the absence (solid circles) and in the presence of the pentapeptide at 20 µM (open circles), 40 µM (solid squares ), or 60 µM (open squares).

Molecular weight estimation

When acid PNGase from embryos of which stage ranging from 10 to 28 was applied to Sephadex S-300 gel filtration, the enzymatic activity eluted as a single peak (result not shown). The apparent molecular weight of this activity was estimated to be 150 K. Because of the technical difficulties involved in isolating blastoderms from embryos in amounts sufficient for biochemical experiments, we were not able to estimate the molecular size of the neutral PNGase activity.

Discussion

In this study we have shown the presence of two different PNGase activities in O.latipes embryos. The first, an acid PNGase M is an activity we previously identified in O.latipes embryos. The second is a neutral PNGase M, identified for the first time in O.latipes blastoderms. These findings demonstrate that multiple forms of PNGase are involved in as yet unknown physiological processes. Taken together, the developmental stage-dependent profiles of the appearance of these enzyme activities, the accumulation of free glycan chains originally attached to specific precursor glycoproteins, and formation of the de-N-glycosylated protein(s) all suggest that each PNGase may act on specific substrates, and thus play distinct physiological roles during embryogenesis. Acid PNGase M has its maximum activity at pH of 3.5-4.0, suggesting that the enzyme is of lysosomal origin. To our knowledge, no other animal cell-derived PNGase with an acid optimum pH has been reported, although the plant PNGase A from almond meal has an acidic optimum pH (Takahashi and Nishibe, 1978). Acid PNGase M can act on several glycoprotein substrates, including O.latipes H- and L-hyosophorin, and glycophosphoproteins (MU-1 and MU-2) (Iwasaki et al., 1992), whereas other glycoproteins such as fetuin and ovalbumin, are poor substrates. Because fetuin glycopeptide II and ovalbumin-derived glycopeptides were readily hydrolyzed by acid PNGase M, it is postulated that the resistance of N-linked sugar chains of native fetuin and ovalbumin to hydrolysis is due to steric hindrance by these polypeptide chains. Due to their high sugar content (80-90%) (Kitajima et al., 1989) and high degree of phosphorylation (Iwasaki et al., 1992), hyosophorins and glycophosphoproteins may adopt extended conformations in aqueous solution, allowing acid PNGase M to gain access to the glycoasparagine linkages.

Notably, the acid PNGase activity is competitively inhibited by one of the reaction products, a de-N-glycosylated peptide, with a relatively low KI (13 µM for Leu-Ala-Asp-CmCys-Ser derived from fetuin glycopeptide II). This reaction was carried out at pH 3.7 where the [beta]-carboxyl group of aspartic acid residue would be partially protonated. Thus, an aspartic acid-protonated peptide might be recognized as a homologue of glycosylasparagine linkage by the acid PNGase M. Free aspartate does not inhibit, so it seems necessary that the [alpha]-amino- and/or [alpha]-carboxyl groups of aspartic acid are in peptide bond for the binding of peptide to the enzyme. This interpretation is supported by the result that the asparagine-oligosaccharide is not hydrolyzed by acid PNGase M (Table IV). The inhibitory effect of the PNGase-generated peptides on acid PNGase M activity might play an important role in regulating the degradation rate of target glycoproteins at different stages during development.

The existence of neutral PNGase M in the O.latipes embryo was demonstrated using isolated blastoderms as source of the enzyme. PNGase activity in the blastoderms was identified by identification of two reaction products from fetuin [14C]glycopeptide II. The first was a free oligosaccharide containing di-N-acetylchitobiosyl structure at the reducing terminus. The second was a free peptide containing aspartic acid residue, derived from glycosylated asparagine. Formation of [14C]Me2Leu as a minor product may be due to an endogenous peptidase. Neutral PNGase M has an optimum pH between 7.5 and 8.5. In contrast to acid PNGase M, the activity was not inhibited by either of the reaction products, but was inhibited by SH-modifying reagents. Further, activity of the neutral PNGase M was detectable between 32 cells and the early gastrula stage.

A group of PNGases with neutral optimum pH values were shown to be ubiquitously expressed in various animal cells, including mouse fibroblast L-929 cells (Suzuki et al., 1994a), several mouse organs (Kitajima et al., 1995) and hen oviduct (Suzuki et al., 1997). In our recent studies, we have found that L-929 cells produce two different neutral PNGases activities at different stages of cell growth. The first is expressed in the log phase of cultured L-929 cells (Chang et al., 1997). The second is expressed when these cells reach confluency. The enzymatic properties of these PNGases were found to be distinctly different from each other. It has been postulated that the hen oviduct PNGase is most likely responsible for quality control of de novo synthesized proteins inside the cell (Suzuki et al., 1998a,b). These enzymes share common properties, such as optimum neutral pH values and loss of activity in the presence of SH-modifying reagents. While principally localized in the cytosol, they are also expressed in the ER (Suzuki et al., 1997). The neutral PNGase M from O.latipes has similarities to these mammalian cell PNGases, at least with respect to their optimum pH values and their sensitivity to SH-modifying reagents, although subcellular localization of the enzymes remains to be elucidated. The biological role of the de-N-glycosylated L-hyosophorin and free glycans that appear in fish embryos as developmentally regulated products of PNGase catalysis also remains to be elucidated.

Previously we found free N-glycans that were presumably produced from hyosophorin, hyosophorin-type free glycans, by the action of a PNGase at stages between fertilization and the early gastrula of O.latipes (Seko et al., 1991a). The same type of free glycans were also found in the embryos of P. olivaceus (Seko et al., 1989). Both acid and neutral PNGase M activities were identified in O.latipes embryos during the early development. However, the neutral PNGase M rather than the acid PNGase M appears to be responsible for the production of the free hyosophorin-type glycan chains from the following two reasons. First, high specific activity of the neutral PNGase M was observed in blastoderms of the embryos at stages when hyosophorin-type free glycans accumulated. Second, no acid PNGase M activity was detected in the embryos at the same developmental stages, although the acid PNGase M activity became prominently expressed at stages later than early gastrula. Thus, neutral PNGase M in the blastoderm is postulated to be a key enzyme that may control the biological function(s) of L-hyosophorin during early development. Our previous studies suggested that L-hyosophorin may be involved in regulation of cell-to-cell interactions, based on the multivalency of its glycan side chains (Taguchi et al., 1994), the species-specific glycan structures and its localization in the perivitelline space surrounding the embryo (Kitajima et al., 1989; Inoue and Inoue, 1997).

On the other hand, another type of free glycans, glycophosphoprotein-type free glycans, which are released from glycophosphoprotein or its putative precursor protein, vitellogenin (Iwasaki et al., 1992), is known to be present in developing embryos. Glycophosphoprotein is a phosvitin-related, major yolk polyphosphorylated glycoprotein (Iwasaki et al., 1992) that appears to be metabolized as nutrients for the growth of embryos during the yolk-absorptive stages. Murakami et al., (1990) showed that degradation of yolk phosphoproteins occurs in O.latipes embryos at stages after gastrulation. It has also been shown that in yolk-absorptive stages, several acidic enzymes are responsible for degradations of yolk materials not only in O.latipes but also in sea urchin (Mallya et al., 1992; Murakami et al., 1992). In this study, acid PNGase M, which has its optimum activity at pH 3.5-4.5, was shown to express its maximal activity at the yolk-absorptive stage, and to be capable of cleaving the glycan chains from the glycophosphoproteins. Taken together, these findings suggest that acid PNGase M is responsible for the production of the glycophosphoprotein-type free glycans and, accordingly, may contribute to the degradation and the absorption of glycophosphoprotein by the developing embryos. It is important to note, however, that the stage when the glycophosphoprotein-type free glycans accumulate varies between different fish species. In O.latipes, this type of glycan chain can be isolated only from the embryos after blastula stages (Kitajima et al., unpublished obser-vations). In contrast, in P.altivelis and T.hakonensis, the glycans are already produced during maturation of the eggs before fertilization (Inoue et al., 1989; Ishii et al., 1989). In this study, we have shown that an acid PNGase is also present in the O.latipes ovaries during maturation. It will now be important to determine if this activity is the same enzyme that is expressed during early development, and to determine the target glycoproteins for the acid PNGase M in ovaries.

Materials and methods

O.latipes embryos

Embryos were collected from adult females early in the morning and, after adhesive threads were cut off, they were incubated in Yamamoto's solution (0.75% NaCl, 0.02% KCl, 0.02% CaCl2, 0.002% NaHCO3 (pH 7.3)) at 27.5°C for 2 h (stage 5; 8 cells), 3 h (stage 7; 32 cells), 5 h (stage 9; late morula), 7.5 h (stage 11; middle blastula), 10.5 h (stage 13; early gastrula), 21 h (stage 18; formation of optic buds), 40 h (stage 25; circulation in vitelline veins; 18-19 segments), or 50 h (stage 28; black pigmentation of eyes; 30 segments). Developmental stages for the embryos were determined morphologically according to the classification by Yamamoto, (1975).

Isolation of the blastoderms from O.latipes embryos

Eggs of O.latipes are telolecithal and, after fertilization, develop by way of discoidal cleavage, thereby forming a blastoderm at the animal pole side (Figure 6, left). About 10,000 blastoderms were surgically excised from the embryos at the blastula stage by using a disposable scalpel and forceps under a stereoscopic microscope. The scalpel was used to pierce at the vegetal pole of the embryos and the fertilization envelope was grasped carefully by the forceps. After the fertilization envelope was cut equatorially with the scalpel, the blastoderm was squeezed out of the residual yolk. Blastoderms were in a semicircular shape immediately after isolation and then became spherical within 30 min, as shown in Figure 6 (right). Isolated blastoderms were washed three times with Yamamoto's solution and kept at -80°C until use.


Figure 6. O.latipes embryos at the middle blastula stage (left) and blastoderms isolated surgically from the embryos at the same stage (after 30 min fromthe operation, right). Scale bar, 1 mm.

It should be noted that because isolation of blastoderms from medaka fish embryos required skillfulness none had ever succeeded in their isolation and the result shown in Figure 6 represents the first successful isolation of medaka fish blastoderms. Blastoderms are extremely fragile and particularly after the early gastrula stage it became impossible to isolate blastoderms. Only at stage 11, this kind of work of a remarkable tour de force under a microscope can only be done for morphological studies in developmental biology but never for biochemical studies. Owing to such technical difficulties inherent in isolating blastoderms from embryos further attempts to provide a molecular basis for the neutral PNGase M by determining its chemical and biochemical properties have been frustrated by the limited availability of blastoderms.

Glycoproteins and glycopeptides

O.latipes H-hyosophorin and L-hyosophorin, Asp-Ala-Ala-Ser-Asn(Fuc2.1Man3.0Gal15.8 GlcNAc9.6NeuAc4.7)-Gln-Thr-Val-Ser, were isolated from the ovary and the fertilized eggs, respectively, as previously described (Kitajima et al., 1989). O.latipes glycophosphoproteins (MU-1 and MU-2) were isolated fromthe ovary as previously described (Iwasaki et al., 1992). Fetuin glycopeptide II, Leu-Ala-Asn(Man3Gal3GlcNAc5NeuAc3)-CmCys-Ser, was prepared from fetal bovine serum fetuin (Nacalai Tesque Co., Kyoto, Japan) according to the method of Plummer et al. (1987). Ovalbumin glycopeptides, Glu-Glu-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val-Leu-Hse, were prepared as described by Plummer and Tarentino (1981). Purification of ovalbumin glycopeptides containing high-mannose and hybrid type glycans was performed as described previously (Suzuki et al., 1994a). Ovalbumin GlcNAc-peptide was prepared from the ovalbumin glycopeptides according to the method of Suzuki et al., (1993). Glycoasparagine, Asn(Man5-GlcNAc4) (GP-IIIA), was prepared from ovalbumin as described previously (Nomoto et al., 1992). Stem bromelain glycopeptide, Ala-Arg-Val-Pro-Arg-Asn-Asn(Fuc0.86Xyl1.0Man2.6-GlcNAc2.0)-Glu-Ser-Ser-Met, and its defucosylated and partially dexylosylated sample obtained by mild acid hydrolysis, Ala-Arg-Val-Pro-Arg-Asn-Asn(Xyl0.52Man2.6GlcNAc2.0)-Glu-Ser-Ser-Met, were prepared as described previously (Suzuki et al., 1994a). Human Bence-Jones Wh [lambda] glycoprotein-derived glycopeptide, Ser-Gly-Asn-Thr-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Ala-Glu-
Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-Ser-Ser-Tyr-Thr-Ser-Asn(Fuc0.85-Man3.0Gal2.0GlcNAc4.9NeuAc1.7)-Ser-Thr-Arg, was kindlyprovided by Dr. K.Yamashita (Sasaki Institute, Tokyo).

14C-Labeling of peptide portion of glycopeptides

14C-Methylation of the primary amino groups in the glycopeptides was carried out by reductive methylation using H14CHO (1.6 GBq/mmol, DuPont/NEN) as described previously (Seko et al., 1991b). The [14C]methyl group was introduced into L-hyosophorin and fetuin glycopeptide II at 0.46 mol and 0.58 mol per mol of the parent glycopeptides, respectively.

Chemical analyses

Carbohydrate compositions were determined by gas-liquid chromatography as described previously (Nomoto et al., 1982). The hexose content was estimated by the phenol-sulfuric acid method (Dubois et al., 1956).

Preparation of free glycans and oligopeptides by PNGase F digestion

L-Hyosophorin-derived nonapeptide (Asp-Ala-Ala-Ser-Asp-Gln-Thr-Val-Ser), fetuin glycopeptide II-derived pentapeptide (Leu-Ala-Asp-CmCys-Ser), and their 14C-methylated derivatives were prepared by digestion with 1-10 mU of PNGase F (Takara Shuzo, Shiga, Japan) (Tarentino and Plummer, 1987). The products were purified by Sephadex G-50 gel filtration (1.2 × 69.5 cm) equilibrated and eluted with 0.1 M NaCl and desalted by Sephadex G-25 gel filtration (1.3 × 69 cm) equilibrated and eluted with 5% ethanol.

Partial purification of acid PNGase M

Acid PNGase M was partially purified from O.latipes embryos as previously reported (Seko et al., 1991b).

Identification of neutral PNGase activity in O.latipes blastoderms

Blastoderms isolated from 3,000 embryos were homogenized in 1.8 ml of 50 mM Tris-HCl (pH 8) containing 5 mM EDTA, 0.25 M sucrose, and 360 µg soybean trypsin inhibitor (Sigma) by vortexing. The homogenate was centrifuged at 100,000 × g for 1 h. To the supernatant, 93 nmol of 14C-labeled asialo fetuin glycopeptide II was added and the mixture was incubated at 25°C for 24 h with a drop of toluene. The digest was then subjected to gel filtration on a Sephadex G-50 column (1.2 × 69 cm, equilibrated and eluted with 0.1 M NaCl). Radioactive fractions and the phenol-sulfuric acid-positive fractions, which were more retarded than the intact glycopeptide, were separately pooled and subjected to gel filtration on a Sephadex G-25 column (1.3 × 69 cm; equilibrated and eluted with 5% ethanol). The [14C]peptide eluted in the column Vo was subjected to silica gel TLC (Kieselgel 60, Merck) developed for 2.5 h in ethyl acetate/pyridine/acetic acid/water (5:5:1:3, v/v/v/v), or by paper electrophoresis on Whatman 3MM paper at 13 V/cm for 2.5 h in pyridine/acetic acid/water (5:0.2:95, v/v/v, pH 6.5). Radioactive compounds on the dried chromatograms were visualized with a Bio-Imaging analyzer (Fujix BAS 2000, Japan). The low-molecular-weight and phenol-sulfuric acid positive fractions (free oligosaccharide), obtained by Sephadex G-25 gel filtration, were analyzed for the sugar composition before and after NaBH4 reduction.

Assay for acid PNGase M

Six microliters of the enzyme solution containing 50 mM sodium citrate buffer (pH 3.7), 0.25 M sucrose, and [14C]L-hyosophorin (10,000 c.p.m.) were incubated at 25°C for 1 h and spotted on Whatman 3MM paper. The paper was developed in 1-butanol/ethanol/water (4:2:3, v/v/v) for 1 h. After drying, a 1.2-2.5 cm portion from the origin containing the [14C]peptide was cut off each lane of the paper and the radioactivity determined in an Aloka Liquid Scintillation System LSC-700. One unit of the enzyme activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 µmol of [14C]L-hyosophorin at 25°C in 1 min under the conditions described above.

Assay for neutral PNGase M

Six milliliters of the enzyme solution containing 50 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 1.2 µg of soybean trypsin inhibitor, 5 mM EDTA, and 10,000 c.p.m. of [14C]fetuin glycopeptide II, was incubated at 25°C for 4 h. The amount of the released [14C]peptide was estimated as described above, except that a slit of the paper between 1.2 and 3.0 cm from the origin was analyzed.

Molecular weight estimation

The apparent molecular weight of acid and neutral PNGase M was estimated by Sephacryl S-300 (high resolution grade) gel filtration. The column (1.3 × 92 cm) was equilibrated and eluted with 5 mM Tris-HCl (pH 8)-0.25 M sucrose, 0.1 M NaCl at flow rate of 11 ml/h. Horse spleen ferritin (Mr, 440,000; Sigma), bovine liver catalase (Mr, 210,000; Sigma), bovine serum albumin (Mr, 69,000; Sigma), ovalbumin (Mr, 45,000; Sigma), and soybean trypsin inhibitor (Mr, 20,100; Sigma) were used as molecular weight markers.

PNGase activity in the embryos of O.latipes at various developmental stages

For acid PNGase M activity, 2.5 g (wet weight) of ovaries or the whole embryos at stages 5 (8 cells), 9 (late morula), 13 (early gastrula), 18 (formation of optic buds), 25 (circulation in vitelline veins; 18-19 segments), or 28 (black pigmentation of eyes; 30 segments) were homogenized and fractionated by ammonium sulfate precipitation to remove endogenous L-hyosophorin, as described previously (Seko et al., 1991b). Each precipitate was dissolved in 250 µl of 5 mM Tris-HCl (pH 8), 0.25 M sucrose, 0.1 mg/ml of soybean trypsin inhibitor, and assayed the acid PNGase activity after dialysis against 5 mM Tris-HCl (pH 8), 0.25 M sucrose.

For neutral PNGase M activity, blastoderms isolated from 20 embryos at the stages 7 (32 cells), 9, 11 (middle blastula), or 13 were homogenized in 6 µl of 0.1 M Tris-HCl (pH 7.5), 0.25 M sucrose containing 10,000 c.p.m. of [14C]fetuin glycopeptide II and incubated at 25°C for 4 h.

Substrate specificity of acid PNGase M

To determine the amount of [14C]peptides released by PNGase M from [14C]glycopeptides, the incubation mixture (6 µl) containing 50 mM sodium citrate buffer (pH 3.7), 0.25 M sucrose, 0.18 nmol of [14C]glycopeptides, and 1.2 µU of the enzyme was incubated at 25°C for 20 min, then analyzed by paper chromatography or the paper electrophoresis. Authentic [14C]peptides were prepared by PNGase F or PNGase A (Seikagaku Co., Tokyo, Japan) digestion of [14C]glycopeptides. With the glycoprotein substrates, amounts corresponding to 0.1 µmol of N-linked glycans were used. Appropriate amounts of ovalbumin, fetal bovine serum fetuin (Mr, 48,400), O.latipes H-hyosophorin (Mr, 128,000), O.latipes glycophosphoprotein MU-1 (Mr, 25,000) or MU-2 (Mr, 10,000) were incubated in 300 µl of 50 mM sodium citrate buffer (pH 3.7), 0.25 M sucrose with 0.25 mU of the enzyme at 25°C for 24 h under a drop of toluene. After the addition of 300 ml of the same enzyme, the incubation mixture was further incubated at 25°C for 24 h. Each of the digests was applied to a Sephadex G-50 column (1.2 × 69 cm, equilibrated, and eluted with 0.1 M NaCl) for estimating the amount of free oligosaccharides released.

Kinetics

The Michaelis constant of acid PNGase M for [14C]L-hyosophorin was estimated by measuring initial reaction rates at various concentrations of substrate (12-250 µM) at 25°C for 7 min. The inhibitory effects of L-hyosophorin-derived nonapeptide and fetuin glycopeptide II-derived pentapeptide on acid PNGase M were investigated by following the alteration of the apparent Km for [14C]L-hyosophorin in the presence of the nonapeptide at 40, 100, or 200 µM, or the pentapeptide at 20, 40, or 60 µM.

Acknowledgments

Most of the research reported in this paper was conducted at the University of Tokyo, where this project was supported in part by Grant-in-Aid (to Y.I.) from the Ministry of Education, Science, and Culture of Japan. The authors are grateful to one of the reviewers for his/her valuable suggestions for revision of the original manuscript. This work was also supported by National Science Council Grant NSC 88-2311-B-001-010 (to Y.I.).

Abbreviations

CmCys, S-carboxymethyl-l-cysteine; H-hyosophorin, cortical alveolar-located glycopolyprotein or high-molecular weight hyosophorin; L-hyosophorin, low-molecular weight hyosophorin; MIA, monoiodoacetic acid; PCMB, p-chloromercuribenzoic acid; PMSF, phenylmethanesulfonyl fluoride; PNGase, peptide-N4-(N-acetyl-[beta]-d-glucosaminyl) asparagine amidase (peptide:N-glycanase) (EC 3.5.1.52)

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