Site‐specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion

Site‐specific structural characterization of the glycosylation of human lecithinrcholesterol acyltransferase (LCAT) was carried out using microbore reversed‐phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate‐specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected TV‐linked glycopeptides of LCAT, a di‐O‐linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N‐linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O‐linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O‐linked glycopeptide, each of which contain sialylated galactoseβ13N‐acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N‐linked glycosylation sites at Asn43 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 7766:301–304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.

[1]  M. F. Bean,et al.  Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry , 1993, Protein science : a publication of the Protein Society.

[2]  E. Davidson,et al.  Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. , 1977, The Journal of biological chemistry.

[3]  A. Dell.  Preparation and desorption mass spectrometry of permethyl and peracetyl derivatives of oligosaccharides. , 1990, Methods in enzymology.

[4]  G. Utermann,et al.  Substitution in vitro of lecithin-cholesterol acyltransferase. Analysis of changes in plasma lipoproteins. , 1980, European journal of biochemistry.

[5]  C. Fielding,et al.  Lecithin-cholesterol acyltransferase: effects of mutagenesis at N-linked oligosaccharide attachment sites on acyl acceptor specificity. , 1993, Biochimica et biophysica acta.

[6]  Y. Uchida,et al.  Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. , 1979, Journal of biochemistry.

[7]  J. Elder,et al.  endo-beta-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Carr,et al.  Analysis of proteins and glycoproteins at the picomole level by on-line coupling of microbore high-performance liquid chromatography with flow fast atom bombardment and electrospray mass spectrometry: a comparative evaluation. , 1990, Biomedical & environmental mass spectrometry.

[9]  L. Hood,et al.  High-sensitivity sequencing with a gas-phase sequenator. , 1983, Methods in enzymology.

[10]  S. Michnick,et al.  Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine. , 1989, Biochemistry.

[11]  G. W. Jourdian,et al.  The purification and properties of beta-galactosidase from bovine testes. , 1973, The Journal of biological chemistry.

[12]  M. F. Bean,et al.  Integration of mass spectrometry in analytical biotechnology. , 1991, Analytical chemistry.

[13]  B. Chait,et al.  Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. , 1992, Science.

[14]  Richard D. Smith,et al.  Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry. , 1989, Analytical biochemistry.

[15]  M. Karas,et al.  Matrix-assisted UV-laser desorption/ionization mass spectrometric analysis of monoclonal antibodies for the determination of carbohydrate, conjugated chelator, and conjugated drug content. , 1991, Analytical chemistry.

[16]  W. Lindenmaier,et al.  Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide. , 1989, The Journal of biological chemistry.

[17]  W. Kohr,et al.  Cloning and expression of human lecithin-cholesterol acyltransferase cDNA. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  C. Fielding,et al.  Human plasma lecithin-cholesterol acyltransferase. Characterization of cofactor-dependent phospholipase activity. , 1978, The Journal of biological chemistry.

[19]  C. Fielding,et al.  Effects of inhibitors of N-linked oligosaccharide processing on the secretion, stability, and activity of lecithin:cholesterol acyltransferase. , 1991, Biochemistry.

[20]  M. Mann,et al.  Electrospray ionization for mass spectrometry of large biomolecules. , 1989, Science.

[21]  M F Bean,et al.  Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests. , 1993, Analytical chemistry.

[22]  V. Wray,et al.  Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells. , 1993, European journal of biochemistry.

[23]  F. Maley,et al.  The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H. , 1974, The Journal of biological chemistry.

[24]  C. G. Edmonds,et al.  New developments in biochemical mass spectrometry: electrospray ionization. , 1990, Analytical chemistry.

[25]  S. Martin,et al.  Site-specific carbohydrate identification in recombinant proteins using MALD-TOF MS. , 1993, Analytical chemistry.

[26]  C W Sutton,et al.  Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry. , 1994, Analytical biochemistry.

[27]  S. Wilson,et al.  The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. , 1988, The Journal of biological chemistry.

[28]  A. Burlingame,et al.  Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry, complementary mobile phases, and sequential exoglycosidase digestion , 1994, Journal of the American Society for Mass Spectrometry.

[29]  S. Kornfeld,et al.  Comparative aspects of glycoprotein structure. , 1976, Annual review of biochemistry.

[30]  M. Gelb,et al.  Human plasma lecithin-cholesterol acyltransferase. Inhibition of the phospholipase A2-like activity by sn-2-difluoroketone phosphatidylcholine analogues. , 1989, The Journal of biological chemistry.

[31]  A. Scanu,et al.  Isolation, properties, and mechanism of in vitro action of lecithin: cholesterol acyltransferase from human plasma. , 1979, The Journal of biological chemistry.

[32]  E. Bause,et al.  Primary structural requirements for N‐glycosylation of peptides in rat liver , 1979, FEBS letters.

[33]  B. Shushan,et al.  Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry. , 1991, Analytical chemistry.

[34]  J. Henion,et al.  Characterization of N-linked oligosaccharides by electrospray and tandem mass spectrometry. , 1992, Analytical chemistry.

[35]  A. Lacko,et al.  Characterization of lecithin-cholesterol acyltransferase from human plasma. 3. Chemical properties of the enzyme. , 1983, Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire.

[36]  R W Sweet,et al.  Protein and carbohydrate structural analysis of a recombinant soluble CD4 receptor by mass spectrometry. , 1989, The Journal of biological chemistry.

[37]  A. Gotto,et al.  Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme. , 1987, The Journal of biological chemistry.

[38]  A. Kobata.  Use of endo- and exoglycosidases for structural studies of glycoconjugates. , 1979, Analytical biochemistry.

[39]  H. Lu,et al.  Glycosidase digestion, electrophoresis and chromatographic analysis of recombinant human granulocyte colony-stimulating factor glycoforms produced in Chinese hamster ovary cells. , 1993, Journal of chromatography.

[40]  Y. Lee.  [89] α-Mannosidase, β-glucosidase, and β-galactosidase from sweet almond emulsin , 1972 .

[41]  R. B. Trimble,et al.  Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1, endo F2, and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans. , 1991, The Journal of biological chemistry.

[42]  L. Warren,et al.  Bound Carbohydrates in Nature: The carbohydrates of glycoproteins , 1994 .

[43]  M. Karas,et al.  Ultraviolet–laser desorption/ionization mass spectrometry of femtomolar amounts of large proteins , 1989 .

[44]  S. Carr,et al.  Carbohydrate mapping by mass spectrometry: a novel method for identifying attachment sites of Asn-linked sugars in glycoproteins. , 1986, Analytical biochemistry.

[45]  A. Burlingame,et al.  Strategies for the Characterization of Carbohydrates from Glycoproteins by Mass Spectrometry , 1994 .

[46]  J. Albers,et al.  Purification and characterization of human plasma lecithin:cholesterol acyltransferase. , 1976, Biochemistry.

[47]  P. Subbaiah,et al.  Low density lipoprotein-activated lysolecithin acylation by human plasma lecithin-cholesterol acyltransferase. Identity of lysolecithin acyltransferase and lecithin-cholesterol acyltransferase. , 1980, The Journal of biological chemistry.

[48]  A. Lacko,et al.  Characterization of lecithin:cholesterol acyltransferase from human plasma: II. Physical properties of the enzyme. , 1983, Archives of biochemistry and biophysics.

[49]  G von Heijne,et al.  Amino acid distributions around O-linked glycosylation sites. , 1991, The Biochemical journal.

[50]  Stephen A. Martin,et al.  Mass spectrometric determination of the amino acid sequence of peptides and proteins , 1987 .

[51]  A. Burlingame,et al.  n-Alkyl p-aminobenzoates as derivatizing agents in the isolation, separation, and characterization of submicrogram quantities of oligosaccharides by liquid secondary ion mass spectrometry. , 1991, Analytical biochemistry.