Sage Journals: Discover world-class research

Abstract

We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils. Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl α-2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules. Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules. High iron diamine-thiocarbohydrazide-silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids. Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD. These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II.

Keywords

keratan sulfate acidic glycoconjugates eosinophil-specific granules mouse

Eosinophils contain an abundance of specific granules. These granules exhibit acidophilia in late eosinophils of bone marrow and mature eosinophils of peripheral blood. This acidophilia is mainly due to highly basic granule proteins: eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin in the matrix, and major basic protein in crystalloids (Venge 1993). In contrast, staining with basic dyes at low pH has demonstrated the presence of strongly acidic substances in some eosinophil-specific granules (Dunn and Spicer 1969). Since then, studies of acidic glycoconjugates in specific granules have been performed using various staining methods such as high iron diamine (HID) (Dunn and Spicer 1969), antimonate-osmium tetroxide (Hardin and Spicer 1970), dialyzed iron (Hardin and Spicer 1971), HID-thiocar-bohydrazide-silver proteinate (HID-TCH-SP) (Parmley et al. 1982), low iron diamine-TCH-SP (Parmley et al. 1982), and cationic colloidal gold (Yang et al. 1998). In combination with enzyme degradation, these staining methods have demonstrated the presence of sulfated glycoconjugates in eosinophil-specific granules. The biochemical composition of sulfated glycosaminoglycans (GAGs) from human eosinophils has been shown to be chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate (Metcalfe et al. 1982). The other type of acidic residue, sialyl residues, are relatively large, hydrophilic, and acidic molecules that have a considerable influence on the physicochemical features of the substances to which they are bound (Schauer et al. 1995). They are widely distributed in tissues and play important biological roles (Rosenberg 1995). This study was undertaken to examine whether sialyl glycoconjugates are expressed in murine eosinophil-specific granules.

Materials and Methods

Mice

Ten-week-old female (C57BL/6 x DAB/2) F₁-BDF₁, C57BL/6, and BALB/c mice were purchased from Japan SLC (Shizuoka, Japan).

Tissue Preparation

Bone marrow samples were fixed in half-strength Karnovsky's fixative composed of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PBS). They were then dehydrated in a graded ethanol series at −35C. After infiltration with Lowicryl K4M resin (Polysciences; Washington, PA), the specimens were placed in capsules with some fresh resin. Polymerization was performed initially at −35C for 24 hr and then at 20C for another 24 hr under indirect, diffuse UV irradiation using a low-temperature polymerization apparatus TTP 010 (Balzer Union; Lichtenstein). Ultrathin sections were cut with an Ultracut N (Reichert-Jung; Vienna, Austria) ultramicrotome and collected on Formvar-coated gold grids.

High Iron Diamine-Thiocarbohydrazide-Silver Proteinate Physical Development (HID-TCH-SP-PD) Staining (Table 1)

The specimens were stained for 18 hr at room temperature (RT) with a high iron diamine solution consisting of 1.6 ml 40% FeCl₃, 120 mg N,N-dimethyl-m-phenylene-diamine-(HCl)₂ (Sigma Chemical; St Louis, MO), and 20 mg N,N-dimethyl-p-phenylene-diamine-HCl (Sigma) in 50 ml distilled water, final pH 1.4. The grids were immersed in 1% thiocarbohydrazide (Sigma) in 20% acetic acid solution for 60 min at RT, washed serially with 10% and 5% acetic acid, and finally washed with distilled water. The sections were then stained with 1% silver proteinate (Merck; Darmstadt, Germany) solution for 60 min and finally developed with a physical developer IntenSE M kit (Amersham International; Poole, UK) for 3 min at RT. The sections were finally counterstained with 2% aqueous uranyl acetate for 3 min.

Lectin Staining (Table 1)

Ultrathin sections were incubated in PBS containing 1% BSA (PBS-BSA) for 30 min at RT. The sections were then incubated in biotinylated Maackia amurensis lectin II (MAL II) or Sambucus nigra (SAN) (Vector Laboratories; Burlingame, CA) lectin dissolved in PBS-BSA overnight at 4C. After two washes with PBS, the sections were incubated in 15-nm colloidal gold-labeled streptavidin (1:40) dissolved in PBS-BSA. The sections were then washed twice with PBS, stained with 2% uranyl acetate for 3 min, and then with Millonig's lead for 1 min at RT.

Enzyme Degradation (Table 1)

For neuraminidase treatment, ultrathin sections were treated with 1 U/ml Clostridium neuraminidase (Sigma) in 0.1 M acetate buffer (pH 5.0) with 0.04 M CaCl₂ for 18 hr at 37C according to the method of Roth et al. (1984). For keratanase II treatment, ultrathin sections were treated with 0.1 U/ml keratanase II (Seikagaku; Tokyo, Japan) in 0.05 M sodium acetate buffer (pH 6.5) for 18 hr at 37C according to the method of Ito et al. (1996). For keratanase treatment, ultrathin sections were treated with 2 U/ml keratanase (Pseudomonas sp.) (Seikagaku) in 0.05 M Tris-HCl buffer (pH 7.4) for 18 hr at 37C according to a modification of the method of Nakazawa et al. (1975) and the manufacturer's protocol. For chondroitinase ABC (Seikagaku) treatment, ultrathin sections were treated with 2 U/ml protease-free chondroitinase ABC in 0.2 M Tris-HCl (pH 8.0) for 18 hr at 37C according to the method of Goode et al. (1991). Control sections were incubated in the same buffer alone for the same period. Afterwards, sections were washed in distilled water three times for 10 min and then stained with MAL II lectin or HID-TCH-SP-PD as described above.

Electron Microscopy

Electron microscopic observation was performed with a Hitachi H-7000 transmission electron microscope at 75 kV accelerating voltage.

Morphometry

Morphometric analysis of colloidal gold labeling on the eosinophil-specific granules was performed using electron micrographs (magnification x 15,000). We measured the areas of the granules and colloidal gold particles using NIH Image (Version 1.58) imaging software from the National Institutes of Health (Bethesda, MD) on a personal computer and calculated the labeling intensity of colloidal gold on the granules. The granules cut in the middle were randomly chosen and measured in approximately 50 cells.

Statistical Analysis

Differences among groups were analyzed statistically by the Mann-Whitney test.

Results

Expression of Sialyl α-2,3 Galactose Residues in Murine Eosinophil-specific Granules

To examine whether eosinophil-specific granules express sialyl glycoconjugates, we performed lectin cytochemistry with MAL II lectin specific for sialyl α-2,3 galactose or SAN lectin specific for sialyl α-2,6 galactose. MAL II lectin positively labeled immature and mature specific granules (Figures 1A and 1B). The labeling was higher in mature granules than in immature granules (Table 2). The positive labeling was completely eliminated by pretreatment with Clostridium neuraminidase, not only in the specific granules but also other areas of eosinophils (Figure 1C). No labeling was observed with SAN lectin (not shown).

Figure 1

Expression of sialyl residues in murine eosinophil-specific granules with and without enzyme treatment. (A) MAL II lectin staining of eosinophil-specific granules. (B) A higher magnification of A. (C) MAL II lectin staining after pretreatment with Clostridium neuraminidase. Almost no labeling is observed. (D) MAL II lectin staining after pretreatment with keratanase II. Almost no labeling is seen except in cytoplasm. Bars = 0.5 μm.

Localization of Sialyl Residues in Eosinophil-specific Granules to Keratan Sulfate

To examine whether sialyl residues are linked to keratan sulfate, the sole proteoglycan that contains sialic acids (Toda and Seno 1970; Choi and Meyer 1975), MAL II lectin staining was performed after treatment with neuraminidase-free keratanase II. As shown in Figure 1D, the enzyme treatment completely eliminated the labeling of MAL II lectin in the specific granules.

HID-TCH-SP-PD staining showed various degrees of positive labeling in crystalloid-free immature granules (Figure 2A), consistent with the pattern of positive labeling with HID-TCH-SP in human and rabbit eosinophil specific granules reported by Parmley et al. (1982). The positive HID-TCH-SP-PD staining was slightly decreased by pretreatment with keratanase (Figure 2B), or keratanase II (not shown). This staining was not completely eliminated but was greatly decreased by pretreatment with chondroitinase ABC specific for chondroitin sulfate and dermatan sulfate (Figure 2C). Finally, staining was completely eliminated by a combination of chondroitinase ABC and keratanase (Figure 2D), or a combination of chondroitinase ABC and keratanase II (not shown). These results suggest that keratan sulfate in the specific granules is of the corneal type (Figure 3), because corneal-type keratan sulfate I is sensitive to both keratanase and keratanase II, whereas keratan sulfate II is sensitive to keratanase II but resistant to keratanase.

Table 1

Specificity of stainings and glycosidases a

Stainings/glycosidases	Specificity	Reference
HID-TCH-SP-PD	Sulfated glycoconjugates	Murata et al. 1992
MAL II lectin	Sialyl α-2,3 galactose residues	Wang and Cummings 1988
SNA lectin	Sialyl α-2,6 galactose residues	Shibuya et al. 1987
Clostridium neuraminidase	Sialyl α-2,3,sialyl α-2,6, sialyl α-2,8 residues	Corfield et al. 1981
Chondroitinase ABC	Chondroitin sulfate, dermatan sulfate	Oike et al. 1982
Keratanase	β1-4 galactosidic linkages	Nakazawa et al. 1989
Keratanase II	β1-3 glucosaminidic linkages	Nakazawa et al. 1989

^aHID-TCH-SP-PD, high iron diamine-thiocarbohydrazide-silver proteinate physical development; MAL II, Maackia amurensis lectin II; SNA, Sambucus nigra.

Figure 2

Localization of sialyl residues in eosinophil-specific granules with and without enzyme treatment. (A) HID-TCH-SP-PD staining of eosinophil-specific granules. Various degrees of labeling in crystalloid-free immature granules are seen. (B) HID-TCH-SP-PD staining after pretreatment with keratanase. Much labeling is still observed. (C) HID-TCH-SP-PD staining after pretreatment with chondroitinase ABC. Labeling is greatly decreased (D) HID-TCH-SP-PD staining after pretreatment with chondroitinase ABC and keratanase. No labeling is observed. Bar = 0.5 μm.

Discussion

The present studies, using a combination of lectin cytochemistry with MAL II specific for sialyl α-2,3 galactose residues and enzyme degradation methods, demonstrated that sialyl residues are expressed in eosinophil-specific granules. The sialyl residues were shown to be linked to sulfated proteoglycan using a combination of HID-TCH-SP-PD staining and enzyme degradation. These results confirm the presence of keratan sulfate in eosinophil-specific granules.

The nucleotide sugar CMP-N-acetylneuraminic acid is synthesized in the nucleus (Kean 1991), and glycosylation reactions occur in the lumen of the endoplasmic reticulum and Golgi apparatus (Hirschberg and Snider 1987; Verbert et al. 1987). This explains the difference in MAL II lectin cytochemical staining between pretreatment with neuraminidase and with keratanase II. Neuraminidase was able to eliminate all positive labeling of MAL II in other areas as well as the specific granules, whereas keratanase II eliminated the positive labeling of MAL II that was restricted to specific granules.

Keratan sulfate proteoglycans are classified into two types on the basis of their linkage to protein (Stuhlsatz et al. 1989) (Figure 3). Keratan sulfate I is linked to protein by GlcNAc → asparagine and is found in the cornea (Seno et al. 1965). The second type, keratan sulfate II, is linked to protein by O-glycosidic linkages between N-acetylgalactosamine and serine or threonine, and is found in cartilage and intervertebral disks (Anderson et al. 1964). These two types of keratan sulfate can be distinguished by their sensitivity to keratanase and keratanase II (Nakazawa et al. 1989). Keratan sulfate I is sensitive to both keratanases, whereas keratan sulfate II is sensitive to keratanase II but resistant to keratanase. Keratanase cleaves the internal β1-4 galactosidic linkages in keratan sulfate and requires at least one N-acetyl-glucosamine 6-sulfate residue adjacent to the galactose residue participating in the galactosidic linkage. Keratanase II cleaves β1-3 glucosaminidic linkages to galactose, as well as those in keratan sulfates to galactose-6-sulfate (Nakazawa et al. 1989). Keratanase contains a small amount of neuraminidase, but keratanase II is negative for contaminating neuraminidase. We next examined the sensitivity to these two enzymes using HID-TCH-SP-PD staining.

Eosinophil-specific Granules Are Sensitive to Both Keratanase and Keratanase II, Suggesting that They Contain Keratan Sulfate I

The distribution of keratan sulfates and their other functions are not clearly understood. Keratan sulfate I (corneal keratan sulfate) appears to play an important role in maintaining the transparency of the tissue in addition to the common role of proteoglycans, which control the concentration gradient of water in the stroma (Nakazawa et al. 1989). In comparison with keratan sulfate, sialic acids have been analyzed much more extensively. They occur mainly as terminal sugar residues, conferring chemical and biological diversity to glycoconjugates. One of the functions of sialic acids is related to their relatively large hydrophilic and acidic features, which exert physicochemical effects on glycoconjugates and their environment (Schauer et al. 1995). Their other function is in molecular and cellular recognition by masking galactose residues to prevent biological recognition or by making sialyl residues serve as recognition sites (Schauer et al. 1995). Sialyl residues expressed in eosinophil-specific granules may cooperate with sulfated residues in the safe storage of highly basic granule proteins by neutralizing them with their acidity, and enzymes by inactivating their activity.

The present study demonstrated that chondroitinase ABC and keratanase (or keratanase II) are necessary for completely eliminating the positive labeling produced by HID-TCH-SP-PD. Parmley et al. (1982) reported that testicular hyaluronidase partially removed the HID-TCH-SP-stained material, and suggested the presence of chondroitin 4- and 6-sulfate and other sulfated glycoconjugates. Metcalfe et al. (1982) demonstrated biochemically that the composition of sulfated glycosaminoglycans from human eosinophils was 70–81% chondroitin 4-sulfate, 9–12% chondroitin 6-sulfate, and 5–12% dermatan sulfate. Because chondroitinase ABC is specific for chondroitin sulfate and dermatan sulfate (Oike et al. 1982), the results of the present study are consistent with their findings. Furthermore, we clearly demonstrated that the remaining sulfated GAG in eosinophils is keratan sulfate.

Figure 3

Carbohydrate structure of keratan sulfates (I and II). Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; Fuc, fucose; Asn, asparagine; NeuAc, sialic acid; Ser, serine; Thr, threonine. From Stuhlsatz et al. (1989), with permission.

HID-TCH-SP-PD staining showed that murine eosinophil-specific granules have a staining pattern similar to those of human and rabbit, and also can be divided into five developmental stages. Parmley et al. (1982) stained human and rabbit developing eosinophils with HID-TCH-SP and divided their specific granules into five developmental stages: precursor granules, labeled in the center of the granule; immature granules Type I, labeled in both the granule center and periphery; immature granules Type II, labeled in the granule perpihery; immature granules Type III, no labeling and lack of a crystalloid core; mature granules, no labeling but a crystalloid core present. Lack of labeling with HID-TCH-SP(-PD) is consistent with the decreased distribution of radiosulfate in mature specific granules (Parmley et al. 1982).

Popken-Harris et al. (1998) reported that human eosinophil-specific granules in the process of condensation possess the proform of major basic protein (proMBP) (Barker et al. 1988; McGrogan et al. 1988) around the periphery and have MBP in the central area. The cDNA for MBP encodes a prepromolecule with a total length of 222 amino acids. PreproMBP includes a secretory leader of 15 amino acids, an acidic propiece of 90 amino acids (Shikata et al. 1993; Pop-ken-Harris et al. 1995), and a basic MBP portion of 117 amino acids. The propiece has an estimated pI of 3.9 and confers an overall acidic charge on proMBP. Popken-Harris et al. obtained eosinophils with specific granules by culturing umbilical cord stem cells with interleukin 5. However, the granules were in the process of condensation and did not become the mature specific granules containing a crystalloid core. These authors concluded that the conversion of proMBP to MBP occurs in the granule, and their results support the hypothesis that the propiece of proMBP functions to protect maturing eosinophils from the toxic MBP during granule processing. Furthermore, they pointed out that a lack of HID-TCH-SP labeling in immature granules Type III and mature granules is consistent with the almost total lack of labeling of proMBP in the condensing central area. However, their finding raises the question of whether toxic MBP that loses the propiece in the central area is harmful to specific granules.

Table 2

Quantification of MAL II labeling in eosinophil-specific granules a

Mice	Numbers of colloidal gold particles/μm² area of granule
BDF₁	Immature granule	Mature granule
		Matrix	Crystalloid
	4.198 ± 10.123 ∗	12.358 ± 16.978
		5.923 ± 22.108	23.440 ± 25.927

^aAreas of granule were measured using NIH image. Data indicate means ± SD of 400 granules measured.

^∗ p>0.0001 by Mann-Whitney test against mature granules.

The present study demonstrated that, in contrast to HID-TCH-SP-PD staining, sialyl residues are positive in mature specific granules (Figures 1A and 1B) and that their expression is higher in mature than in immature specific granules (Table 2). HID-TCH-SP-PD method detects sulfated residues by forming electrostatic linkage with ionized sulfated residues in the tissue section. Absence of labeling does not always suggest lack of sulfated residues but may also suggest the presence of cationic substances that neutralize the negative charges of sulfated residues. This concept is consistent with the results of low but positive labeling with autoradiography using ³⁵SO₄ ^2- and lack of labeling with HID-TCH-SP-PD staining in mature granules reported by Parmley et al. (1982). On the other hand, MAL II lectin binds sialyl α-2,3 galactose residues not with electrostatic strength but with more specific affinity. Therefore, sialyl residues can be detected by MAL II lectin as long as the residues are present in the tissue section. A biochemical study has demonstrated that GAGs may form complexes with lysosomal enzymes and inactivate them (Avila and Convit 1976; Avila 1978). Corresponding findings of masking of acid phosphatase reactivity have been reported in mature eosinophil granules (Wetzel et al. 1967; Bass et al. 1981). The present results of the positive labeling of sialyl residues and the negative labeling of HID-TCH-SP-PD in mature granules also support this concept. Sialyl residues would play a similar role to other sulfated GAGs in specific granules. Therefore, they appear to share with other sulfated GAGs the role of complexing with highly basic granule proteins and inactivating them during the maturation of specific granules. The presence of another acidic GAG, keratan sulfate, suggests that the functions of eosinophil-specific granules are mediated by many substances, including keratan sulfate and other sulfated GAGs.

The sialyl residues can be detected by a MAL II lectin in neutral buffer in contrast to sulfate residues in very acidic buffer, suggesting that the molecules can be isolated without denaturation for further analysis with techniques of molecular biology. Eosinophils play an important role in allergy and inflammatory diseases by releasing their granule proteins. Clarification of the mechanism for safe storage of highly basic granule proteins and their release will help the development of therapy for allergy or eosinophil-related diseases. Analysis focusing on these sialyl molecules will contribute to the purpose.

Footnotes

Acknowledgements

Supported in part by the Kodama Foundation for Research in Medical Science.

We wish to thank Mr S. Nonaka for technical assistance and Mr S. Imamura for secretarial assistance.

References

Anderson

Seno

Sampson

Riley

Hoffman

Meyer

(1964) Threonine and serine linkages in mucopolysaccharides and glycoproteins. J Biol Chem 239:PC2716–2717.

Avila

(1978) The influence of the type of sulphate bond and degree of sulphation of glycosaminoglycans on their interaction with lysosomal enzymes. Biochem J 171:489–491.

Avila

Convit

(1976) Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro: a model of control of leucocytic lysosomal activity. Biochem J 160:129–136.

Barker

Gleich

Pease

(1988) Acidic precursor revealed in human eosinophil granule major basic protein cDNA. J Exp Med 168:1493–1498.

Bass

Lewis

Szejda

Cowley

McCall

(1981) Activation of lysosomal acid phosphatase of eosinophil leukocytes. Lab Invest 44:403–409.

Choi

Meyer

(1975) The structure of keratan sulphates from various sources. Biochem J 153:543–553.

Corfield

Veh

Wember

Michalski

Schauer

(1981) The release of N-acetyl- and N-glycolloyl-neuraminic acids from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidase. Biochem J 197:293–299.

Dunn

Spicer

(1969) Histochemical demonstration of sulfated mucosubstances and cationic proteins in human granulocytes and platelets. J Histochem Cytochem 17:668–674.

Goode

Shires

Aparicio

Davison

(1991) Cationic colloidal gold−a novel marker for the demonstration of glomerular polyanion status in routine renal biopsies. Nephrol Dial Transplant 6:923–930.

10.

Hardin

Spicer

(1970) An ultrastructural study of human eosinophil granules: maturational stages and pyroantimonate reactive cation. Am J Anat 128:283–310.

11.

Hardin

Spicer

(1971) Ultrastructural localization of dialyzed iron-reactive mucosubstance in rabbit heterophils, basophils, and eosinophils. J Cell Biol 48:368–386.

12.

Hirschberg

Snider

(1987) Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 56:63–87.

13.

Ito

Yokota

Nagaike

Morimura

Hatake

Tanaka

Matsunaga

(1996) Simultaneous expression of keratan sulphate epitope (a sulphated poly-N-acetyllactosamine) and blood group ABH antigens in papillary carcinomas of the human thyroid gland. Histochem J 28:613–623.

14.

Kean

(1991) Sialic acid activation. Glycobiology 1:441–447.

15.

McGrogan

Simonsen

Scott

Griffith

Ellis

Kennedy

Campanelli

Nathan

Gabay

(1988) Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein. J Exp Med 168:2295–2308.

16.

Metcalfe

Litvin

Wasserman

(1982) The isolation, identification and characterization of sulfated glycosaminoglycans synthesized in vitro by human eosinophils. Biochim Biophys Acta 715:196–204.

17.

Murata

Tsuyama

Ihida

Kashio

Kawano

Zeng

(1992) Sulfated glycoconjugates demonstrated in combination with high iron diaminethiocarbohydrazide-silver proteinate and silver acetate physical development. J Electron Microsc 41:14–20.

18.

Nakazawa

Ito

Yamagata

Suzuki

(1989) Substrate specificity of keratan sulphate-degrading enzymes (endo-β-galactosidase, keratanase and keratanase II) from microorganisms. In Greiling

Scott

, eds. Keratan Sulphate: Chemistry, Biology, Chemical Pathology. London, Biochemical Society, 99–110.

19.

Nakazawa

Suzuki

(1975) Sequential degradation of keratan sulfate by bacterial enzymes and purification of a sulfatase in the enzymatic system. J Biol Chem 250:905–911.

20.

Oike

Kimata

Shinomura

Suzuki

Takahashi

Tanabe

(1982) A mapping technique for probing the structure of proteoglycan core molecules. J Biol Chem 257:9751–9758.

21.

Parmley

Takagi

Spicer

Thrasher

Denys

(1982) Ultrastructural visualization of complex carbohydrates in eosinophilic leukocytes. Am J Anat 165:53–67.

22.

Popken-Harris

Checkel

Loegering

Madden

Springett

Kephart

Gleich

(1998) Regulation and processing of a precursor form of eosinophil granule major basic protein (ProMBP) in differentiating eosinophils. Blood 92:623–631.

23.

Popken-Harris

McGrogan

Loegering

Checkel

Kubo

Thomas

Moy

Sottrup-Jensen

Snable

Kikuchi

Gleich

(1995) Expression, purification, and characterization of the recombinant proform of eosinophil granule major basic protein. J Immunol 155:1472–1480.

24.

Rosenberg

(1995) Biology of the Sialic Acids. New York, Plenum Press.

25.

Roth

Lucocq

Charest

(1984) Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity tecnique with the use of fetuin-gold complexes. J Histochem Cytochem 32:1167–1176.

26.

Schauer

Kelm

Reuter

Roggentin

Shaw

(1995) Biochemistry and role of sialic acids. In Rosenberg

, ed. Biology of the Sialic acids. New York, Plenum Press, 7–67.

27.

Seno

Meyer

Anderson

Hoffman

(1965) Variations in keratosulfates. J Biol Chem 240:1005–1010.

28.

Shibuya

Goldstein

Broekaert

Nsimba-Lubaki

Peeters

Peumans

(1987) The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2–6)Gal/GalNAc sequence. J Biol Chem 262:1596–1601.

29.

Shikata

Hayashi

Yoshimatsu

Ohya

Seto

Fukushima

Yoshida

(1993) Pro-major basic protein has three types of sugar chains at the pro-portion. Biochim Biophys Acta 1163:243–249.

30.

Stuhlsatz

Keller

Becker

Oeben

Lennartz

Fischer

Greiling

(1989) Structure of keratan sulphate proteoglycan: core protein, linkage regions, carbohydrate chains. In Greiling

Scott

, eds. Keratan Sulphate: Chemistry, Biology, Chemical Pathology. London, Biochemical Society, 1–11.

31.

Toda

Seno

(1970) Sialic acid in the keratan sulfate fraction from whole cartilage. Biochim Biophys Acta 208:227–235.

32.

Venge

(1993) Human eosinophil granule proteins: structure, function and release. In Smith

Cook

, eds. Immunopharmacology of Eosinophils. London, Academic Press, 43–55.

33.

Verbert

Cacan

Cecchelli

(1987) Membrane transport of sugar donors to the glycosylation sites. Biochimie 69:91–99.

34.

Wang

Cummings

(1988) The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues. J Biol Chem 254:2786–2792.

35.

Wetzel

Spicer

Horn

(1967) Fine structural localization of acid alkaline phosphatases in cells of rabbit blood and bone marrow. J Histochem Cytochem 15:311–334.

36.

Yang

D-H

Tsuyama

Ohmori

Murata

(1998) Sulphated glycosaminoglycans in guinea pig eosinophils studied by means of cationic colloidal gold. Histochem J 30:1–6.

Keratan Sulfate Glycosaminoglycans in Murine Eosinophil-specific Granules

Abstract

Keywords

Materials and Methods

Mice

Tissue Preparation

High Iron Diamine-Thiocarbohydrazide-Silver Proteinate Physical Development (HID-TCH-SP-PD) Staining (Table 1)

Lectin Staining (Table 1)

Enzyme Degradation (Table 1)

Electron Microscopy

Morphometry

Statistical Analysis

Results

Expression of Sialyl α-2,3 Galactose Residues in Murine Eosinophil-specific Granules

Localization of Sialyl Residues in Eosinophil-specific Granules to Keratan Sulfate

Discussion

Eosinophil-specific Granules Are Sensitive to Both Keratanase and Keratanase II, Suggesting that They Contain Keratan Sulfate I

Footnotes

Acknowledgements

References