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Isolation and partial characterization of a novel keratan sulfate proteoglycan from metamorphosing bonefish (Albula) larvae

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Abstract

Proteoglycans (PGs) were isolated from leptocephalous larvae of the bonefish (Albula sp.), which were in the early stages of metamorphosis, using both associative and dissociative conditions in the presence of protease inhibitors. The procedure was rapid and resulted in an extraction efficiency of 75% (associative) and 85–90% (dissociative). The majority of co-extracted protein could be effectively separated from the PGs by utilizing either Sepharose CL-2B or CL-6B gel chromatography. Sepharose CL-2B chromatography of extracted PGs after treatment with bacterial keratan sulfate-endo-β-galactosidase (keratanase) showed that most of the high molecular weight (M r) carbohydrate was degraded. Free keratan sulfate (KS) chains were prepared from whole-larva extracts (which also contain small amounts of chondroitin sulfate) by both chondroitinase ABC treatment and ethanol fractionation. Sepharose CL-6B chromatography under dissociative conditions showed that larval KS chains were much larger (M r∼55,000) than those from cornea. These chains tended to aggregate when chromatographed under associative conditions. Larval KS was degraded by keratanase and resistant to chondroitinase, ABC and testicular hyaluronidase. Differences were also noted in the oligosaccharides produced by keratanase treatment of the two preparations. However, biochemical composition of larval and corneal KS was similar.

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Abbreviations

KS:

keratan sulfate

CS:

chondroitin sulfate

Gal:

galactose

GlcN:

glucosamine

GlcNAc:

N-acetylglucosamine

EDTA:

ethylenediaminetetraacetic acid

PG:

proteoglycan

GAG:

glycosaminoglycan

SDS:

sodium dodecyl sulfate

PAGE:

polyacrylamide-gel electrophoresis

TLC:

thin-layer chromatography

V o :

void volume

V t :

total volume

M r :

molecular weight

U:

units

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  1. Edward Pfeiler

    Present address: Department of Biology, University of Puerto Rico, 00708, Mayaguez, Puerto Rico

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  1. Departamento de Ciencias Marinas, Instituto Tecnológico y de Estudios Superiores de Monterrey, Guaymas, Apartado Postal 484, 85400, Sonora, Mexico

    Edward Pfeiler

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Pfeiler, E. Isolation and partial characterization of a novel keratan sulfate proteoglycan from metamorphosing bonefish (Albula) larvae. Fish Physiol Biochem 4, 175–187 (1988). https://doi.org/10.1007/BF01871744

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