Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein
2000
The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-α1–6-mannose-β1–4-N-acetylglucosamine-N-acetylglucosamine(α1–6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. Copyright © 2000 John Wiley & Sons, Ltd.
Abbreviations:
CSAct
cerebroside sulfate activator protein
C8
octyl hydrocarbon chain
dHex
deoxyhexose
ESIMS
electrospray ionization mass spectrometry
5-, 2-, 1- and 0-CHO
5, 2, 1 and 0-oligosaccharide containing
Fuc
fucose
GlcNAc
N-acetylglucosamine
(Hex)n
n hexose units
(HexNAc)n
nN-acetylhexose units
HPLC
high-performance liquid chromatography
HUA
human urine CSAct
LC/MS
liquid chromatography/electrospray ionization mass spectrometry
Man
mannose
MS/MS
tandem mass spectrometry
NP
normal-phase
MALDI
matrix-assisted laser desorption/ionization mass spectrometry
P
parent ion
PNGase
peptide N-glycosidase
PKA
pig kidney CSAct
RP
reversed-phase
TFA
trifluoroacetic acid
TMAPA
trimethyl-p-aminophenylammonium chloride
W/A/F
water–acetonitrile–formic acid (50 : 50 : 0.1, v/v).
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