Keratan Sulfate Glycosaminoglycans in Murine Eosinophil-specific Granules

Abstract

SUMMARYWe 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 ␣-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.

Intracellular proteoglycans

“…In addition, basophils and mucosal mast cells [46] have been shown to contain the disaccharide 2-O-sulphated IdoA-4-O-sulphated GalNAc (CS-diB), as shown in Table 1. The presence of both intracellular keratan sulphate [47] and hyaluronan [18,21] has also been documented.…”

Capillary electrophoresis for total glycosaminoglycan analysis

“…Keratanase cleaves the internal β1–4 galactosidic linkages in KS and generally affords oligosaccharides. In contrast, keratanase II cleaves β1–3 glucosaminidic linkages to galactose, as well as those in KS to galactose-6-sulfate, releasing monosulfated or disulfated disaccharides from KS [11]. …”

Localization of Sialyl Glycoconjugates in Eosinophil-Specific Granules after Degranulation Stimuli

“…In previous studies, we demonstrated that sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan [5], which are the sole proteoglycan-containing sialic acids [8]. A biochemical study demonstrated that GAGs may form complexes with lysosomal enzymes, inactivating their enzyme activities [9, 10].…”

“…Studies using various staining methods have demonstrated that the acidic substances are sulfated [3, 4]and sialyl glycoconjugates [5]. Sulfated residues were labeled by high iron diamine-thiocarbohydrazide-silver proteinate-physical development (HID-TCH-SP-PD) staining [6].…”