of biological chemistry 268, pp. by for u.s.a. structural ... · the journal 0 1993 by the american...

THE JOURNAL 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

OF BIOLOGICAL CHEMISTRY Vol. 268, No. 1, Issue of January 5, pp. 113-126,1993 Printed in U.S.A.

Structural Elucidation of a Variety of GalNAc-containing N-Linked Oligosaccharides from Human Urinary Kallidinogenase”

(Received for publication, July 21, 1992)

Noboru Tomiya$, Juichi Awaya$, Masayasu KuronoS, Hiroyuki HanzawaQ, Ichio ShimadaQ, Yoji Aratas, Tomoaki Yoshidaq, and Noriko Takahashill** From the SMie Research Laboratory, Sanwa Kagaku Kenkyusho Co. Ltd., 363 Shiozaki, Hokusei-cho, Inabe-gun, Mie 51 1-04, Japan, the §Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo 113, Japan, the TNagoya University Medical School, Showa-ku, Nago.ya 464, Japan, and IIGl.ycoLab, Nakano Central Research Institute, Nakuno Vinegar Co., Ltd., 2-6 Nakamura-cho, Handa, Ailhi 475, Japan

.. .

Fifteen different structures of terminal GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase have been identified. These N-linked oligosaccharides were mostly neutral, because sialic acid content was lower than 0.13 mol of sialic acid/mol of sugar chain, and sulfate was not detected. The oligosaccharides were released from pepsin-digested protein by glycoamidase A (from almond) digestion. The reducing ends of the oligosaccharide chains were aminated with a fluorescent reagent, 2-aminopyridine. The resulting mixture of pyridylamino derivatives of the oligosaccharides were separated by high performance liquid chromatography on an ODS-silica column, and 15 oligosaccharides were isolated. The structure of each oligosaccharide fraction was analyzed by two- dimensional sugar mapping, component sugar analysis, high resolution proton nuclear magnetic resonance and methylation analysis. It was found that each N-linked oligosaccharide associated with human urinary kallidinogenase contains unsubstituted GalNAc residues at the nonreducing terminal. These 15 oligosaccharides include 5 biantennary, 7 triantennary, and 3 tetraantennary oligosaccharides.

Human urinary kallidinogenase is a serine protease that liberates lysyl-bradykinin, a vasoactive decapeptide, from kin- inogens. Recently, the complete amino acid sequence of human urinary kallidinogenase was reported and three N-gly- cosylation sites were identified, Asn-78, Asn-84, and Asn-141 (1,28,29). The structures of the N-linked carbohydrate chains of human urinary kallidinogenase were unknown, so we un- dertook an analysis of these structures.

We have developed a two-dimensional (2-D)’ sugar mapping technique by HPLC, which is a convenient and powerful method for structural analysis of minute quantities of N- linked oligosaccharides (2). More than 170 pyridyl aminated

* The costs of Publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom all correspondence should be addressed: GlycoLab, Nakano Central Research Institute, Nakano Vinegar Co., Ltd., 2-6 Nakamura-cho, Handa, Aichi 475, Japan. Tel.: 81-569-24-5114; Fax:

The abbreviations used are: 2-D, two-dimensional; PA, pyridyl aminated HPLC, high performance liquid chromatography; ’H NMR, proton nuclear magnetic resonance; GC-MS, gas chromatography-mass spectroscopy; Gal, D-galactose; Man, D-mannose; Fuc, L- fucose; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galac- tosamine; HexNAc, N-acetylhexosamine,

81-569-24-5028.

(PA) oligosaccharides as standard were chromatographed on two different HPLC columns (ODs-silica and amide-silica), and the elution positions of standard oligosaccharides on both columns were plotted as the coordinates on a 2-D sugar map (30). The unknown structure of oligosaccharides can be esti- mated by comparing the elution positions (expressed in glucose units) of a sample with those of the standard oligosaccharides. Using this method, we can easily characterize the unknown structure of oligosaccharides, if they are of the usual type.

In this report, we describe a group of oligosaccharides with unique structures which were obtained from human urinary kallidinogenase. Since these oligosaccharides are released by glycoamidase A digestion, they are all N-linked oligosaccharides. However, on the 2-D sugar map, the elution positions of these oligosaccharides did not coincide with those of any standard oligosaccharides, unless direct @-N-acetylhexosaminidase digestion was run. That is, the oligosaccharides derived from human urinary kallidinogenase were shown to bear unique structures. Recently, N-linked oligosaccharides containing N-acetylgalactosamine (GalNAc) have been found in a number of glycoproteins (3-8, 16, 17, 31). As to the tripeptide, Pro-Xaa-Arg/Lys, required for recognition by GalNAc transferase, we will discuss this briefly.

EXPERIMENTAL PROCEDURES

Enzymes

Glycoamidase A from almond (obtainable glycopeptidase A), 0- galactosidase and fl-N-acetylhexosaminidase from jack bean were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). a-L-Fucosidase from bovine kidney was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Pepsin was purchased from Sigma.

Reference Oligosaccharides

The PA-isomalto oligosaccharides (4-25 glucose units) were purchased from Nakano Vinegar Co. The structures of a series of reference compounds are shown in Table I (code No. are cited from Ref. 2) and prepared as follows. The pyridylamino derivatives of triman- nosy1 core oligosaccharides without fucose (000.1), with fucose (010.1), and monoantennary oligosaccharides (100.3 and 110.3) were prepared as described previously (9). The pyridylamino derivatives of biantennary (210.4), triantennary (310.8 and 310.18), and tetraantennary (410.16) N-acetyllactosamine-type oligosaccharides were prepared from recombinant erythropoietin (10). The other monoantennary (110.9) and biantennary (210.11) oligosaccharides were prepared by P-N-acetylhexosaminidase digestion of the oligosaccharides 410.3 and 410.9, respectively, which were obtained from porcine pancreatic kallidinogenase (11). Three unusual oligosaccharides (210.14, 210.15, and 310.19) were determined in the process of this research.

113

114 Terminal GalNAc-containing N-Linked Oligosaccharides

TABLE I Structures and code numbers of the pyridylaminated oligosaccharides used in this study

Code No. Structures

000.1

010.1

100.3

110.3

110.8

110.9

210.4

210.11

Mana6\

1 Manp4GlcNAcp4GlcNAc Mana3

Mana6\ Fuca6

1 1 Manp4GlcNAcp4GlcNAc

Mana3

Galp4GlcNAcp2Mana6 \ 1 Manp4GlcNAcp4GlcNAc

Mana3

Galp4GlcNAcp2Mana6 \ 1 1

Fuca6

Manp4GlcNAcp4GlcNAc

Mana3

Galp4GlcNAcp6 \ Mana6\

Fuca6 1

1 Manp4GlcNAcp4GlcNA Mana3

Fuca6 1

Galp4GlcNAcp4 Manp4GlcNAcpIGlcNAc \ I Mana3

Galp4GlcNAcp2Mana6 \ 1 1

Fuca6

Manp4GlcNAcp4GlcNAc

Galp4GlcNAcp2Mana3

GalpIGlcNAcp6 \ /Mana6\

Fuca6 1

Galp4GlcNAcp2 Manp4GlcNAcp4GlcNA 1 Mana3

Galp4GlcNAcp6 \

\ I Mana6\

Fuca6 1

210.14 Galp4GlcNAcp4 Manp4GlcNAcpBGlcNAC

Mana3 .___

Terminal GalNAc-containing N-Linked Oligosaccharides 115 TABLE I-continued

code No. Structures

IMana6\ Fuca6 GalpBGlcNAcp2 I GalsBGlcNAcp4 r anp4GlcNAcp4GlCNAc \

210.15

Manas

310.8

310.18

310.19

410.16

Galp4GlcNAcp2Mana6 \ I \ I

Fuca6

Galp4GlcNAcp4 Manp4GlcNAcp4GlcNAc

/Mana3 GalpBGlcNAcp2

GalpBGlcNAcp6 \ IMana6\ Fuca6

I Galp4GlcNAcp2 Manp4GlcNAcp4GlcNAc I Galp4GlcNAcf32Mana3

Galp4GlcNAcp6 \ Fuca6

Galp4GlcNAcp2 I Galp4GlcNAcp4

anp4GlcNAcp4GlCNAc

\ Mana3

Galp4GlcNAcp6 \

IMana6\ Fuca6 Galp4GlcNAcs2 I Galp4GlcNAcp4

anp4GlcNAcp4GlcNAc

\

Galp4GlcNAcp2

Other Chemicals

The following materials were purchased from the sources indicated Sephadex G-15, Pharmacia LKB Biotechnology Inc. (Uppsala, Swe- den); Bio-Gel P-4 (200-400 mesh), Bio-Rad sodium cyanoborohydride, Aldrich Chemical Co.; 2-aminopyridine, Wako Pure Chemical Industries (Osaka, Japan).

Release of Oligosaccharides from Human Urinary Kallidinogenase by Glycoamidase A Digestion

Pyridylumination of Oligosaccharides Oligosaccharides obtained from human urinary kallidinogenase

were reductively aminated with a fluorescent reagent, 2-aminopyridine, by the method of Hase et al. (12) with a slight modification as follows.

Reagents-2-Aminopyridine solution consisted of 1.0 g of recrys- tallized 2-aminopyridine dissolved in 0.76 rnl of concentrated (12 N) hydrochloric acid. Reducing reagent consisted of 10 mg of sodium cyanoborohydride dissolved in 20 pi of the 2-aminopyridine solution and 25 p1 of water.

Human urinary kallidinogenase purified from fresh human urine Procedures-1) Place the well-dried sample (about 200 nmol) in a was obtained from JCR Pharmaceuticals Co., Ltd. (Kobe, Japan). Reacti-Vial (Pierce Chemical Co.), add 50 pl of the 2-aminopyridine Oligosaccharides were released by sequential digestion with pepsin solution and mix. 2) Heat the sample at 100 "C for 15 min. 3) Add 3 and glycoamidase A (9) from 60 mg (about 2 @mol) of human urinary pl of the reducing reagent above. 4) Heat the sample at 90 "C for 15 kallidinogenase. The oligosaccharide fraction was collected by gel h. 5) Dilute the sample with 200-300 p1 of water. 6) Purify the PA- filtration on a Bio-Gel P-4 column. oligosaccharides by gel filtration on a Sephadex G-15 column (25 ml)


using 10 mM NH,HCOs as an eluant. By this procedure, about 4 pmol

charides. of PA-oligosaccharides were obtained from about 5 pmol of oligosac-

Isolation of PA-oligosaccharides by Reverse-phase HPLC PA-oligosaccharides derived from human urinary kallidinogenase

were separated on reverse-phase HPLC using a Shimadzu LC-6A chromatograph with a Shimpack CLC-ODS column (0.6 X 15 cm, Shimadzu, Japan). Elution was performed a t a flow rate of 0.8 ml/ min at 55 "C using 10 mM sodium phosphate, pH 3.8 (solvent A), and 10 mM sodium phosphate, 0.5% 1-butanol, pH 3.8 (solvent B). The column was equilibrated with a 95:5 mixture of solvents A and B. After a mixture of PA-oligosaccharides was injected onto the column, the ratio of the solvents was increased over 120 min with a linear gradient to 5050, then held for 40 min. PA-oligosaccharides were detected by fluorescence using excitation and emission wavelengths of 320 and 400 nm, respectively. Each oligosaccharide fraction that corresponded to peaks on the ODS column chromatogram was collected and evaporated to dryness in vacuo.

Structural Characterization of PA-oligosaccharides by 2-0 Sugar Mapping

PA-oligosaccharides which had been isolated as described above were separately subjected to HPLC on two types of columns (2). (i) The first method was reverse-phase HPLC with a Shimpack CLC- ODS column. The column was equilibrated and run at a flow rate of 1 ml/min with a 4:l mixture (by volume) of solvents A and B (described above). After injecting a sample (1-10 pl) onto the column, the ratio of solvent B to solvent A was increased to 50:50 with a linear gradient over 60 min. (ii) The second method was size fractionation HPLC with a TSK-GEL Amide-80 column (0.46 X 25 cm, Tosoh, Japan). Elution was performed at a flow rate of 1.0 ml/min at 40 'C using a 35:65 mixture of 0.52 M acetic acid/triethylamine buffer, pH 7.3, and acetonitrile (solvent C) and a 50:50 mixture of 0.52 M acetic acidltriethylamine, pH 7.3, acetonitrile (solvent D). T h e column was equilibrated with solvent C. After a sample (1-10 r l ) was injected onto the column, a linear gradient was used to obtain 100% solvent D in 50 min. PA-oligosaccharides were detected in both systems by fluorescence as described above.

The elution positions of the sample oligosaccharides on the two column chromatograms were plotted as coordinates (expressed in numbers of glucose unit) on the 2-D sugar map (2).

Exoglycosidase Digestion of PA-oligosaccharides The exoglycosidase digestion procedure of PA-oligosaccharides is

as follows (2). About 0.1-1 nmol of each PA-oligosaccharide was incubated with jack bean &galactosidase (5 milliunits) in 15 pl of 0.1 M citrate-phosphate buffer, pH 4.1, jack bean P-N-acetylhexosaminidase (20 milliunits) in 15 p1 of 0.1 M citrate-phosphate buffer, pH 5.0, and a-L-fucosidase from bovine kidney (20 milliunits) in 15 p1 of 0.1 M acetate buffer, pH 4.5, at 37 "C for 15 h.

Determination of the Sugars Released from PA-oligosaccharides by Exoglycosidase Digestion

The monosaccharides released from PA-oligosaccharides by exoglycosidase digestion were separated by isocratic elution with 16 mM NaOH on a CarboPac PA-1 column (4.6 X 250-mm, Dionex) with a flow rate of 0.8 ml/min. NaOH (300 mM) was added to the post- column effluent with a Dionex anion micro membrane suppressor (AMMS-I). The monosaccharide was monitored with a pulsed am- perometric detector using a gold working electrode and triple-pulse amperometry (13). The following pulse potentials and durations were used for detection of monosaccharides: El = 0.05 V (t l = 360 ms); E2 = 0.80 V (tz = 120 ms); Es = -0.60 V (t3 = 420 ms). The response time of the pulsed amperometry detector was set to 3 S.

' H NMR Measurements 'H NMR measurement was carried out as described previously

(14). Prior to NMR measurement, each PA-oligosaccharide (about 20-100 nmol) isolated by HPLC was desalted by gel filtration on a Sephadex G-15 column. Samples were dissolved in 99.8% %O, lyophilized, and dissolved again in 99.8% 'HzO. 'H NMR measurements were made on a Bruker AM-400 spectrometer operating at 400 MHz in the Fourier-transform mode at 23 (or 24 "C) and 60 "C. Chemical shifts are relative to internal sodium 2,2-dimethyl-2-sila- pentane-5-sulfonate.

GC-MS of Methylated Alditol Acetates Each PA-oligosaccharide (about 50 nmol) isolated by HPLC was

permethylated with finely powdered NaOH and methyl iodide by the method of Ciucanu and Kerek (15). The methylated alditol acetates in the hydrolysates of the permethylated oligosaccharides were analyzed by GC-MS using a SP2380 column (0.53 mm x 30 m; Supelco Inc.) which was linked to a JMS-DX303 mass spectrometer (Japan Electron Opticus Laboratory). The column temperature was increased from 160 to 210 "C at a rate of 2 "C/min. The flow of carrier gas (helium) was set at 12.5 kilopascal of head pressure. Alditol acetates were quantified at m/z = 43 from the mass chromatogram.

RESULTS AND DISCUSSION

Preparation of Oligosaccharides-Carbohydrate composi- tion analyses showed that human urinary kallidinogenase contained 2.9, 5.0, 1.4, 6.8, 4.7, and 0.4 (mol/mol protein) of Gal, Man, Fuc, GlcNAc, GalNAc, and sialic acid, respectively. The content of sialic acid per N-linked sugar chain is calculated to be lower than 0.13, since human urinary kallidinogenase has three N-linked sugar chains and additional 0- linked sugar chains (1, 28, 29). Furthermore, sulfate was not detected in human urinary kallidinogenase after hydrolysis with 6 N HC1 at 100 "C for 20 h (data not shown). These results indicate that most of sugar chains of human urinary kallidinogenase are neutral. The present structural study was, therefore, focused on the neutral moiety of the oligosaccharides. Sialic acid that occurred in minor quantity was com- pletely removed from human urinary kallidinogenase during pepsin digestion at pH 2, 37 "C for 48 h. About 80% of the carbohydrate chains of human urinary kallidinogenase were released by digestion with glycoamidase A. The reducing ends of the oligosaccharides were substituted by the pyridyl amino group. The yield of the modification was about 80% and the reaction proceeded regardless of size or structures of oligosaccharides.

Structural Characterization of PA-oligosaccharides by a Combination of HPLC and Exoglycosidase Digestion-As shown in Fig. 1, the PA-oligosaccharides derived from human urinary kallidinogenase were separated into about 20 peaks on ODs-silica column chromatogram. Fifteen peaks labeled b, f, g, h, i, 1, m, n, p, q, r, s, t, u, and v were collected separately, and subjected to size fractionation HPLC using an amide-silica column. The coordinates of elution position of each oligosaccharide on the ODS- and amide-silica column chromatograms (expressed in numbers of glucose units) were plotted on a 2-D sugar map constructed with standard oligosaccharides (Fig. 2). The elution positions of the sample oligosaccharides were compared with those of standard oligosaccharides which included 170 different compounds. Oli- gosaccharides b-v did not coincide with any standard oligosaccharide on the 2-D sugar map (data not shown), indicating that all of these oligosaccharides have novel or unusual structures. As shown in Fig. 2, however, after sequential digestion with p-galactosidase and p-N-acetylhexosaminidase, the elution position of oligosaccharides b and f shifted to a position identical to that of trimannosyl core oligosaccharide 000.1. The elution positions of the other oligosaccharides shifted to a position identical with that of fucose-containing trimannosyl core oligosaccharide 010.1. These results indicate that the oligosaccharides derived from human urinary kallidinogenase are N-acetyllactosamine-type oligosaccharides and have the same core structure.

Mann6 1Fucn6 \ I

/ Man@4GlcNAc@4GlcNAcfi

Mann3

FIG. 1. Elution profile of PA-oligosaccharides derived from a human urinary kallidinogenase by ODs-silica column. A mixture of PA- oligosaccharides was injected into a Shim-Pack CLC-ODS column. Elution conditions are described under “Experi- mental Procedures.”

Terminal GalNAc-containing N-Linked Oligosaccharides 117

11

10

5

4

1 I I I I I I ‘ 0 80 100 120 140 160

Elution Time (rnin)

*410.18

Pn 7 9

rn

FIG. 2. Structural analyses of oligosaccharides b-v (Fig. 1) by a combination of sequential digestion with &galactosidase and 8-N-acetylhexosaminidase and 2-D mapping techniques. A portion (about 100 pmol each) of PA-oligosaccharides b-v were digested sequentially with j3-galactosidase and j3-N-acetylhexosaminidase. The elution positions on ODS and amide-silica columns of oligosaccharides b-v and the exoglycosidase digests are plotted on a 2-D sugar map (expressed as glucose unit) with reference compounds 000.1, 010.1, 310.8, 310.18, and 410.16. Arrows indicate the directions of changes of the coordinates of oligosaccharides after digestion with @-galactosidase (e) and 8-N-acetylhexosaminidase (DL Details are described under

7 8 9 1 0 11 12 13 14 ,5 16 17 18 19 “Experimental Procedures.” Structures

ODS-Silica (Glucose Units) of reference compounds are shown in Table I.

FIG. 3. Structural analyses of oligosaccharides b-v (Fig. 1) by a combination of direct B-N-acetylhexosaminidase digestion and 2-D mapping techniques. A portion (about 100 pmol each) of PA-oligosaccharides b-v were directly digested with j3-N-acetylhexosaminidase. The elution positions on ODS and amide-silica columns of oligosaccharides b-v and 0-N-acetylhexosaminidase digests are plotted on a 2-D sugar map (expressed as glucose unit) with reference compounds 000.1, 010.1, 100.3, 110.3, 110.9, 210.11, 210.14, 210.15, and 310.19. Arrows indicate the directions of changes of the coordinates of oligosaccharides after digestion with the enzyme. Details are described under “Experimental Procedures.” Structures of reference compounds are shown in Table I.

10

4

7 8 9 10 11 12 13 14 15 16 17 18 19

ODS-Silica (Glucose Units)


Moreover, most of the sample oligosaccharides were converted to known N-linked oligosaccharides with 0, 1, 2, or 3 Gal- GlcNAc-Man sequences by direct @-N-acetylhexosaminidase digestion (Fig. 3). These data indicate that the oligosaccharides obtained from human urinary kallidinogenase have one or more Gal-GlcNAc-Man branches and N-acetylhexosamine (HexNAc) residues linked to Man. They also indicate that the sample oligosaccharides are different from the usual N- linked oligosaccharides (used as standards in our 2-D sugar map) in the outer HexNAc region.

Monosaccharide Analysis in the Outer Chains of Oligosac- charides b-u-The monosaccharides component linked to trimannosyl core 000.1 of each sample oligosaccharide were determined by high performance chromatography-pulsed am- perometric detection with an anion exchange column, after digestion of PA-oligosaccharides with @-galactosidase, @-N- acetylhexosaminidase, and a-L-fucosidase (Table 11). By a-L- fucosidase digestion, all of the oligosaccharides except b and f have 1 fucose residue. By @-galactosidase digestion, oligosaccharides b, m, h, s, v, and r have 1 galactose residue, oligosaccharides g, t, and q have 2 galactose residues, and oligosaccharide n has 3 galactose residues.

By direct P-N-acetylhexosaminidase digestion, the outer GlcNAc and GalNAc residues were all released from oligosaccharides f, 1, p, i, and u (Table 11) and were all converted into the trimannosyl core (Fig. 3). Therefore, only N-acetylhexo- samines attach to the trimannosyl core in these oligosaccharides. On the other hand, 1 Gal and 1 GlcNAc remained in oligosaccharides b, m, h, s, v, and r after direct @-N-acetylhexosaminidase digestion, and the remaining Gal and GlcNAc residues were released by sequential digestion with @-galactosidase and @-N-acetylhexosaminidase. These data indicate that one GalP4GlcNAc branch is bound to the trimannosyl core in these oligosaccharides. Two GlcNAc and two Gal remained in oligosaccharides g, t, and q after direct @-N- acetylhexosaminidase digestion, and the remaining Gal and GlcNAc residues were released by sequential digestion with P-galactosidase and 0-N-acetylhexosaminidase. These data indicate that two GalB4GlcNAc branches are bound to the trimannosyl core in these oligosaccharides. Three GlcNAcs and 3 Gal residues remained in oligosaccharide n after direct

@-N-acetylhexosaminidase digestion, and these Gal and GlcNAc residues were released by sequential digestion with @-galactosidase and @-N-acetylhexosaminidase. These data indicate that three Galp4GlcNAc branches are bound to the trimannosyl core in oligosaccharide n.N-Acetylgalactosamine residues were found in the outer chains of all 15 oligosaccharides and all GalNAc residues were released with GlcNAc residues by direct @-N-acetylhexosaminidase digestion. These results indicate that GalNAc residues exist in (HexNAc), sequences of the outer chains.

The major peaks, m, p, g, i, v, and r, have been shown to bear GalNAc@4GlcNAc-Man sequences by 'H NMR spectroscopy and methylation analysis as described below. This is also thought to be true for the other minor oligosaccharides. Therefore, it appears that the GalNAc residues exist in GalNAc@4GlcNAc-Man branches in other oligosaccharides. Oligosaccharides b, 1, m, g, s, t, and n have 1 GalNAc residue, oligosaccharides f, p, h, u, v, and q have 2 GalNAc residues, and oligosaccharides i and r have 3 GalNAc residues. These data suggest that each of these oligosaccharide contains one or more GalNAcP4GlcNAc branches attached to the triman- nosy1 core with the number of branches corresponding to the number of GalNAc residues present in the oligosaccharide.

Twelve of the 15 PA-oligosaccharides (all except 1, s, and u) released equal numbers of GalNAc and GlcNAc residues after direct @-N-acetylhexosaminidase digestion. Oligosaccha- rides 1, s, and u released 1 more GlcNAc residue than the number of GalNAc residues after P-N-acetylhexosaminidase treatment. This indicates that these compounds contain unsubstituted GlcNAc residues linked to the trimannosyl core. Thus, the total number of GlcNAc residues in one oligosaccharide corresponds to the number of antenna in that mole- cule. The data in Table I1 show that oligosaccharides b, f, 1, m, and p are biantennary, oligosaccharides g, h, i, s, t, u, and v are triantennary, and n, q, and r are tetraantennary. These observations are consistent with the results obtained from 'H NMR spectroscopy, methylation analysis, and 2D-mapping analysis described below.

'H NMR Analysis of Oligosaccharides m, p, g, i, v, and r- The IH NMR spectrum of the most abundant oligosaccharide p is reproduced in Fig. 4. Fig. 5 shows a comparison of 'H

TABLE I1 Sugars released from PA-oligosaccharides by exoglycosidase digestion

Amount of sugars are expressed in mole per mol of PA-oligosaccharides. Released monosaccharide was analyzed by HPAEC-PAD, after sequential digestion with &galactosidase and P-N-acetylhexosaminidase (a, c); direct @-hexosaminidase digestion (b, d); 0-galactosidase digestion (e); a-L-fucosidase digestion (f) of each PA-oligosaccharide.

GalNAc GlcNAc Gal F U C Oligosaccharide

a b A b , b i C d A(c, d) e f - -

Biantennary b f 1 m P

Triantennary

1 2 1 1 2

0 0 0 0 0

0 0 0 0 0 0 0

2 2 2 2 2

3 3 3 3 3 3 3

I 2 2 1 2

1 2 3 2 1 3 2

1 0 0 1 0

2 1 0 1 2 0 1

1 1 1

Tetraantennary n 1 1 0 4 1 3 3 1 q 2 2 0 4 2 2 2 1 r 3 3 0 4 3 1 1 1


anomeric protons - GalNAc6 GalWS

GN5

M4

II

NAc-CH3 pototonr

, GalNAcS GalNAc6

GN5

GN2 (IGNs snomeic protons -

~ ~ ~ ~ ~ ~ ~ ~ ~ I ~ ~ ~ ~ ~ ~ " ' I ~ ~ ~ ' ~ ' " ' I " ' " " ' ~ I " ' ~ ' ~ ~ ~ ' I " ' " " " I " " " " " ~ ~ ~ - " ~ " ~ ~ ~ ~ ' ~ ~ ~ ' l ~ ~ ~ ~ ~ ~ ~ ~ ' l ~ ~ ~ ~ ~ ~ ' ~ - 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 6.0 5.5 5.0 4.5

6(PPm) FIG. 4. 400 MHz 'H NMR spectrum of PA-oligosaccharide p derived from human urinary kallidinogenase recorded in 'H20

at 23 "C (a) and 60 "C (b ) . The letters in the spectrum refer to the corresponding residues in the structure shown in Table 111.

NMR spectra of oligosaccharides m, p, g, i, v, and r. Chemical shift values for the anomeric protons and the methyl protons of these oligosaccharides along with reference compounds 210.4 (biantennary), 310.8 (triantennary), and 410.16 (tetraantennary) are compiled in Table 111. The reference oligosaccharides are all typical N-acetyllactosamine-type oligosaccharides containing fucose as illustrated in Table I.

In oligosaccharides m, p, g, i, v, and r, signals were observed for the anomeric protons at about 4.495 ppm (from 4.487- 4.498 ppm) (Figs. 4 and 5). Corresponding 'H NMR signals were not observed for any of the reference compounds in which Galp4GlcNAc branches are linked to Man-4 and 4'. Baenziger et al. (4) reported that the chemical shifts of the anomeric protons in terminal GalNAc residues in the GalNAcp4GlcNAc sequence bound to Man-4 and 4' are both 4.516 ppm. Therefore, the NMR signals from 4.487-4.498 ppm were assigned to the anomeric protons for terminal GalNAc-6 and GalNAc-6'. The difference between 4.516 and 4.487-4.498 ppm is not negligible, however, there are no signals in these regions except 4.487-4.498 ppm. Moreover, the results of monosaccharide analysis, methylation analysis, and HPLC analysis indicate the existence of terminal GalNAc residues. The signal for the anomeric proton of Gal-8' is observed in downfield comparison with those of the Gal-6, Gal-6', and Gal-8 residues. Similarly, the signal for the anomeric proton of GalNAc-8' (4.511 ppm and 4.507 ppm in oligosaccharide i and r, respectively) occurred in a downfield shift compared with those of GalNAc-6 and GalNAc-6'. Fur- thermore, the signals for methyl protons of all GalNAc residues were essentially the same (between 2.045 and 2.049 ppm). This is due to the terminal position of GalNAc residues, which are not affected by other residues.

The characteristics of the 'H NMR signals attributable to anomeric protons of GlcNAc-5, GlcNAc-5', and GlcNAc-7' bearing terminal GalNAc residues in place of the Gal residue shifted upfield. Thus, the anomeric proton of GlcNAc-5 shifted from 4.573 ppm (reference compound 210.4) to 4.533 ppm (oligosaccharide m) or to 4.536 ppm (oligosaccharide p); from 4.556 ppm (reference compound 310.8) to 4.548 ppm (oligosaccharide g), 4.540 ppm (oligosaccharide i), and 4.518 ppm (oligosaccharide v); from 4.559 ppm (reference compound

410.16) to 4.521 ppm (oligosaccharide r). Similarly, the anomeric proton for GlcNAc-5' shifted from 4.573 ppm (reference compound 210.4) to 4.536 ppm (oligosaccharide p); from 4.569 ppm (reference compound 310.8) to 4.540 ppm (oligosaccharide i) or 4.518 ppm (oligosaccharide v); and from 4.577 ppm (reference compound 410.16) to 4.535 ppm (oligosaccharide r). Oligosaccharide g contains GlcNAc-5', which contains Gal- 6', therefore, the chemical shift of the anomeric proton for GlcNAc-5' of oligosaccharide g is 4.567 ppm and the value is consistent with that of reference compound 310.8 (4.569 ppm). Similar upfield shift of 'H NMR signals were observed for GlcNAc-7' of oligosaccharides i and r, in comparison with reference compound 410.16.

The signals of the anomeric protons for GlcNAc-7 of both oligosaccharides v and r were 4.535 ppm and the values were consistent with those of reference compounds 310.8 (4.537 ppm) and 410.16 (4.538 pprn). These results indicate that oligosaccharides v and r contain Gal-8. The results of 'H NMR analysis of these oligosaccharides are in complete ac- cord with the proposed structures for these compounds as shown in Table IV.

GC-MS of Methylated Alditol Acetates-The most abundant oligosaccharide p was permethylated and hydrolyzed, reduced, and acetylated according to the method of Ciucanu and Kerek (15). The resulting methylated alditol acetates were analyzed with a gas chromatograph-mass spectrometer. The region of the total ion chromatogram which defines the branching pattern of the trimannosyl core and the nature of the HexNAc constituents is shown in Fig. 6a. All potentially significant peaks were identified from mass chromatograms of diagnostic signals. The partially methylated alditol acetates were then identified from their full mass spectra and comparison with elution times of standards. Peaks of n / z 117 and 159 on mass chromatograms (see Fig. 6, b and c, for the origin of these two ions) were also used to define all candidates for amino sugar components. The mass spectrum for amino sugars are reproduced in Fig. 6, b and c. Peaks eluting at 4.54, 10.39, 17.33, 25.45, and 28.24 min were identified as 2,3,4-tri-O-methyl Fuc, 3,4,6-tri-O-methyl-Man, 2,4-di-O-methyl-Man, 3,4,6-tri- 0-methyl-GalNAc, and 3,6-di-O-methyl-GlcNAc, respectively. Taking into account the presence of a major peak for


FIG. 5. 400 MHz 'H NMR spectra of anomeric protons of PA-oligosaccharides m, p, g, i, v, and r derived human urinary kallidinogenase recorded in aHzO at 24 OC (left) and 60 OC (right). The letters (M3, M4, etc.) in the spectra refer to the corresponding residues in the structure on Table 111. Star indicates impurity.

I 24c I r

J i I

9 1

3,4,6-tri-O-methyl-Man, these results suggest that oligosaccharide p is a biantennary oligosaccharide containing 2 terminal N-acetylgalactosamine residues.

In oligosaccharide m, 3,4,6-tri-O-methyl-Man, 2,4,-di-O- methyl-Man, 3,6-di-O-methyl-GlcNAc, 3,4,6-tri-O-methyl- GalNAc, 2,3,4-tri-O-methyl-Fuc, and 2,3,4,6-tetra-O-methyl- Gal were identified (data not shown). The ratio of terminal GalNAc to terminal Gal determined by comparing the peak areas with standards was found to be about 1:l. These results suggest that oligosaccharide m has biantennary structure and has GalNAc and Gal residues at each terminal position.

In oligosaccharide v, 3,4,6-tri-O-methyl-Man, 3,6-di-0- methyl-Man, 2,4-di-O-methyl-Man, 3,6-di-O-methyl-GlcNAc, 3,4,6-tri-O-methyl-GalNAc, 2,3,4-tri-O-methyl-Fuc, and 2,3,4,6-tetra-O-rnethyl-Gal were identified (data not shown). The ratio of terminal GalNAc to terminal Gal was about 21. These results indicate that oligosaccharide v has a 2,4,2- branched triantennary structure and 2 GalNAc and 1 Gal residue at terminal positions.

Structural Analyses by 2 - 0 Sugar Mapping-A variety of oligosaccharides including the N-acetyllactosamine-type, oli- gomannose-type, and hybrid-type oligosaccharides are clearly separated by the 2-D sugar mapping technique (2). As shown in Fig. 2, not only a biantennary oligosaccharide 210.4, and a

tetraantennary oligosaccharide 410.16, but also two triantennary oligosaccharides, 310.8 and 310.18, are clearly separated on the 2-D sugar map. Therefore, we used the 2-D mapping technique to analyze the structures of individual PA-oligosaccharides obtained from human urinary kallidinogenase.

Biantennary Oligosaccharides-Oligosaccharides b, f, 1, n, and p have 2 GlcNAc residues (Table 11) and all cluster in the vicinity of 210.4 (a typical biantennary oligosaccharide) on the 2-D sugar map (Fig. 2).

Oligosaccharide b consists of 1 galactose, 1 GalNAc, and 2 GlcNAc residues in the outer chains and the trimannosyl core 000.1 (Table 11). The position of oligosaccharide b coincided with that of the a-L-fucosidase digest of oligosaccharide m (see below) on the 2-D sugar map. After direct 0-N-acetylhexosaminidase digestion, the position on the 2-D sugar map shifted to that of 100.3 (Table I and Fig. 3).

Oligosaccharide f has neither fucose nor galactose residues and has 2 GlcNAc and 2 GalNAc residues in the outer chains (Table 11). After direct P-N-acetylhexosaminidase digestion, oligosaccharide f was converted to the trimannosyl core, 000.1 (Fig. 3). The position of oligosaccharide f on the 2-D sugar map coincided with that of the a-L-fucosidase digest of oligosaccharide p (see below).

The position of oligosaccharide 1 on the 2-D sugar map


TABLE I11 Chemical shifts of H-1 protons and methyl protons for the pyridylumim derivatives of oligosaccharides derived

from human urinary knllidinogenuse and reference compounds 8' 7' F

GaI84GlsNAc04, ,, FucaO-0-CH 2

Chemical shifisa in Kallidinogenase oligosaccharides Reference compounds

D R 8-1

m

Man-3 (4. 731)b (4.726) (4.729) Man-4 (5.101) (5.099) (5.104) Nan-4' (4.893) (4.882) (4.841) ClcNAc-5 4.533C 4.536 4.548 ClcNAc-5' 4.558 4.536 CICNAC-7 GlcNAc-7' 4.534

4.567

GalNAc-6 4.491 4.495 4.498 GalNAc-6' 4.495 CalNAc-8' Gal-6 Gal-6' 4.449 Gal-8

4.447

Gal-8' Fuc (4.836) (4.843) (4.828)

4.462

NAc GlcNAc-2 2.062 2.064 2.057 ClcNAc-5 2.026 2.028 2.021 CICNAC-5' 2.026 2.017 2.021 ClcNAc-7 ~- ~~ ." . G~cNAc-7' GalNAc-6 2.045

2.021 2.048

GalNAc-6' 2.046

GalNAc-8' 2.048

C h

i V r 210.4 310.8 410.16

(4.723) (4.718) (4.715) (4.749) (4.738) (4.741) (5.1031 (5.101) (5.101) (5.110) (5.112) (5.122) (4.839) (4.871) (4.830) (4.909) (4.849) (4.850) 4.540 4.518 4.540 4.518

4.521 4.573 4.535 4.573

4.556 4.559 4.569 4.577

4.535 4.535 4.537 4.538 4.511 4.521 4.538 4.495 4.487d 4.493 4.495 4.497d 4.493 4.511 4.507

4.461 4.459 4.456

4.444 4.461 4.459 4.456

4.445 4.459 4.456

(4.833) (4.844) (4.823) (4.848) (4.848) (4.850) 4.470

2.060 2.032

2.064 2.049 2.024

2.075 2.026 2.047

2.010 2.017 2.009 2.037 2.036 2.033 2.043 2.047

2.048 2.026 2.065 2.071 2.033 2.010 2.009

2.048 2.048 2.049 2.048 2.048 2.049 2.048 2.049

F"C 1.163 1.167 1.149 1.165 1.169 1.149 1.178 1.169 1.168

Chemical shifts are expressed in ppm from DSS. 'H NMR spectra were measured in 'Hz0 at 60 "C. 'H NMR spectra were measured in 'Hz0 at 24 "C. Assignments may have to be interchanged. The symbols used are: 0, GlcNAc; 0, GalNAc; I, Gal; +, Man; 0, Fuc.

coincided with that of the /+galactosidase digest of oligosaccharide m. After direct 8-N-acetylhexosaminidase digestion, the position of oligosaccharide 1 shifted on the 2-D sugar map to that of the trimannosyl core with fucose residue 010.1 (Fig. 3). Moreover, the position of the a-L-fucosidase digest of oligosaccharide 1 coincided with that of the P-galactosidase digest of oligosaccharide b on the 2-D sugar map.

The structure of oligosaccharide m was studied by 'H NMR measurement and methylation analysis as described above. After N-acetylhexosaminidase digestion, the position of oligosaccharide m shifted to that of the standard monoantennary oligosaccharide 110.3 (Table I and Fig. 3).

The structure of oligosaccharide p was studied by 'H NMR measurement and methylation analysis. It had no galactose residue. After direct 8-N-acetylhexosaminidase digestion, the position of oligosaccharide p shifted to that of the trimannosyl core 010.1 (Table I) on the 2-D sugar map (Fig. 3).

Triantennary Oligosaccharides-The standard triantennary oligosaccharide 310.18 has GlcNAc~6(GlcNAc~2)Mana6 and GlcNAcp2Mana3 arms and another triantennary standard oligosaccharide 310.8 has GlcNAc@BManaG and

GlcNAcp4(GlcNAcp2)Mana3 arms. The sample oligosaccharides have 3 GlcNAc residues in the outer chains (Table 11) separated into two groups. These groups: (i) g, h, and i and (ii) s, t, u, and v are clustered in the vicinity of the typical triantennary standards 310.18 and 310.8, respectively (Fig. 2). From the position on the 2-D sugar map, group (i) oligosaccharides are thought to contain three branches, GlcNAcpGMana6, GlcNAc@PManaG, and GlcNAcpfLMana3. Group (ii) oligosaccharides are also thought to contain three branches, GlcNAcp2Mana6, GlcNAcb4Mana3, and GlcNAcpZMana3.

The structure of oligosaccharide g was studied by 'H NMR measurement as described above. After @-N-acetylhexosaminidase digestion, the position of oligosaccharide g on the 2-D sugar map shifted most to that of standard oligosaccharide 210.11 (Table I and Fig. 3). Slight contamination of @-galactosidase activity in the commercial jack bean P-N-acetylhexosaminidase solution caused additional degradation of GalPGlcNAcP branches and resulted in small amounts of monoantennary oligosaccharides 110.3,110.8, and the triman- nosy1 core 010.1 (all in Table I).

122 Terminal GalNAc-containing N-Linked Oligosaccharides TABLE IV

Proposed structures, relative quantities, and elution positions from Shim-Pack CLC-ODs and TSK-Gel Amide-80 columns, of 15 GalNAc containim PA-olimsaccharides. *, six structures confirmed bv 'H NMR measurements

No. PA-Oligosaccharides No. of Glc units Relative

quantity ODS Amide-80 (8)

b

f

1

m*

P"

g*

h

i*

Galp4GlcNAcpZMana 7 I Manp4GlcNAcp4GlcNAc

GalNAcp4GlcNAcp2Mana3

GalNAcp4GlcNAcp2Mana 7 I Manp4GlcNAcp4GlcNAc


GlcNAcp2Mana 7 Fuca6 I

I Manp4GlcNAcp4GlcNAc GalNAcp4GlcNAcp2Mana3

Galp4GlcNAcp2Mana 7 Fuca6 I


GalNAcp4GlcNAcp2Mana 7 Fuca6 I


Galp4GlcNAcp 7 I Manay Fuca6

I Galp4GlcNAcp2 Manp4GlcNAcp4GlcNAc I


GalNAcp4GlcNAcp6 \ I Manay Fuca6

I Galp4GlcNAcp2 Manp4GlcNAcp4GlcNAC I


GalNAcp4GlcNAcp 7 I Mana7 Fuca6

I GalNAcp4GlcNAcp2 Manp4GlcNAcp4GlcNAC I GalNAcp4GlcNAcp2Mana3

10.2

10.9

13.0

13.6

14.7

11.0

11.7

12.2

6.4

6.2

6.0

6.9

6.7

8.5

2.9

2.5

1.2

19.7

36.7

3.7

8.1 5.1

7.8 2.8


TABLE IV-continued

No. of Glc units Relative No. PA-Oligosaccharides quantity

ODS Amide-80 ( I $ )

GlcNAcp2Mana Fuca6 i

S Galp4GlcNAcp Manp4GlcNAcp4GlcNAc 16.6 7.3 3.8 7 1 /

GalNAcpBGlcNAcp2

t

U

Galp4GlcNAcp2Mana 7 4 /

Fuca6 1

Galp4GlcNAcp Manp4GlcNAcp4GlcNAc 17.2

I Mana3 GalNAcpQGlcNAcp2

GalNAcp4GlcNAcp2Mana Fuca6

G1cNAcp4\ I 1

ManplGlcNAcp4GlcNAc 18.1

I GalNACp4GlCNAcp2

GalNAcp4GlcNAcp2Mana Fuca6 1

V* Galp4GlcNAcp 4 1 Manp4GlcNAcp4GlcNAc 18.5

l'ana3 GalNAcp4GlcNAcp2

8.2 2.1

6.9 2.2

8.0 10.1

n

Galp4GlcNAcp 7 Galp4GlcNAcp2 IMana6\ Fuca6 1

anp4GlcNAcp4GlcNAc 14.0 9.7 2.9 Galp4GlcNAcpy

I Mana3 GalNAcp4GlcNAcp2

Galp4GlcNAcp 7 GalNAcp4GlcNAcp2 IMana6\ Fuca6

1 q anp4GlcNAcp4GlcNAc 15.1

Galp4GlcNAcpj /l 9.4 2.3

Pana3 GalNAcp4GlcNAcp2

Table IV continues on next page.


TABLE IV-continued

No. of Glc u n i t s Relative No. PA-Oligosaccharides quantity

ODS Amide-80 ( % )

GalNAcp4GlcNAcp

Fuca6 GalNAcp4GlcNAcp2 I

r* anp4GlcNAcp4GlcNAc 15.6 9.0 GalplGlcNAcp4, /"

I GalNAcpGlcNAcp2

2.0

After direct P-N-acetylhexosaminidase digestion, the position of oligosaccharide h on the 2-D sugar map changed to that of monoantennary standard oligosaccharide 110.3 (Table I and Fig. 3). After sequential digestion with P-galactosidase and P-N-acetylhexosaminidase, oligosaccharide h resulted in trimannosyl core structure 010.1.

The structure of oligosaccharide i was studied by 'H NMR measurement as described above. This oligosaccharide contained 3 GalNAc residues (Table 11). Oligosaccharide i is triantennary with a GlcNAc/36(GlcNAc~2)Mana6 arm, therefore, the proposed structure in Table IV is most consistent with these results.

After direct P-N-acetylhexosaminidase digestion, oligosaccharide s had the same elution position on the 2-D sugar map as monoantennary oligosaccharide 110.9 (Table I and Fig. 3). After p-galactosidase digestion, the position of oligosaccharide s was consistent with that of the p-galactosidase digest of oligosaccharide t (Fig. 2).

After direct P-N-acetylhexosaminidase digestion, oligosaccharide t was converted to the size of a biantennary oligosaccharide containing two GalP4GlcNAc branches. Additive degradation of the remaining two arms of the biantennary oligosaccharide resulted in small amounts of two monoantennary oligosaccharides, 110.3 and 110.9, and the trimannosyl core, 010.1, by contaminating @-galactosidase activity (Table I). From these experiments, the resulting structure appears to be an unusual biantennary oligosaccharide 210.15 (Table I). Since the structure of oligosaccharide 210.15 remained after P-N-acetylhexosaminidase digestion of oligosaccharide t, the GalNAcfl4GlcNAc arm is attached to the Mana3 arm through a PI-2 bond.

After p-galactosidase digestion of oligosaccharide v, the resultant oligosaccharide had the position of oligosaccharide u on the 2-D sugar map (Fig. 2). Since oligosaccharide v had only 1 galactose residue (Table 11), the proposed structure of oligosaccharide u is reasonable (Table IV).

After direct P-N-acetylhexosaminidase digestion, the position of oligosaccharide v on the 2-D sugar map shifted to that of standard monoantennary oligosaccharide 110.9 (Table I and Fig. 3). This result was consistent with those obtained from 'H NMR measurements and methylation analysis (described above).

Tetraantennary Oligosaccharides-Oligosaccharides n, q, and r have 4 GlcNAc residues (Table 11) and are clustered in the vicinity of 410.16 (a typical tetraantennary oligosaccharide).

Oligosaccharide n is a tetraantennary oligosaccharide with 3 galactose residues and 1 GalNAc residue in the outer chains

(Table 11). One GalNAc and 1 GlcNAc residue were released by direct P-N-acetylhexosaminidase digestion and its position on the 2-D sugar map shifted primarily to that of an unusual triantennary oligosaccharide 310.19 (Fig. 3). Small amounts of three different biantennary oligosaccharides were also pro- duced due to the contaminating p-galactosidase activity. The positions of these three biantennary oligosaccharides were identical with those of 210.11, 210.14 (see the section describ- ing oligosaccharide q), and 210.15, respectively (Table I). From these experiments, the structure of the unusual triantennary oligosaccharide is thought to be 310.19 (Table I). Except for the proposed structure for oligosaccharide n illustrated in Table IV, there is no other clearly identified oligosaccharide reported to contain these particular biantennary structures.

Oligosaccharide q is also tetraantennary with 2 galactose and 2 GalNAc residues in outer chains (Table 11). Two GalNAc and 2 GlcNAc residues were released by direct P-N- acetylhexosaminidase digestion and resulted primarily in an unusual biantennary oligosaccharide 210.14 (Fig. 3). Further- more, minor amounts of two different monoantennary oligosaccharides, 110.8 and 110.9, appeared in the reaction mixture due to the contaminating P-galactosidase activity. These results indicate that one GalP4GlcNAc sequence is attached to M a w 6 through a Pl-6 linkage and another Galb4GlcNAc sequence is attached to Mana3 through a Pl-4 linkage in the intermediate of this unusual biantennary oligosaccharide. The proposed structure of oligosaccharide q is illustrated in Table IV.

The structure of oligosaccharide r was studied by 'H NMR measurement as described above. This is a tetraantennary oligosaccharide with 1 galactose and 3 GalNAc residues in outer chains (Table 11). This structure was resistant to (3-N- acetylhexosaminidase (20 milliunits/500 pmol substrate). When 10-fold excess enzyme was used, however, the position of oligosaccharide r on the 2-D sugar map shifted to that of monoantennary oligosaccharide 110.9. The proposed structure of oligosaccharide r is illustrated in Table IV.

The proposed structures and the relative quantities of all 15 different oligosaccharides derived from human urinary kallidinogenase are summarized in Table IV. The percentage of each component oligosaccharide (b-v) was calculated by its peak area on the HPLC profile since the relative fluorescence is the same on a molar basis for each component (12). These oligosaccharides have unsubstituted terminal GalNAcs as a common component. Several reports have described N-linked carbohydrate chains containing GalNAc residues. These include: (i) biantennary and hybrid N-linked oligosaccharides


FIG. 6. GC-MS of partially methylated alditol acetates derived from PA-oligosaccharide P. a, total ion chromatogram; peaks marked with X are not sugar derived, since their mass spec- t ra did not contain diagnostic sugar signals; b and c, mass spectra of peaks 25.45 and 28.24, respectively.

m C a

U m

c b m c n

W u)

0 Q

2 c z W +- n

W C 0 Q

d 8 c c 0

n

10.39

4.54 17.33

28.24

0 5 10 15 20 25 30 min

1

Peak 25.45

CHDOAc

CHNMeAc 1 I I 159

l j / Peak 28.24

containing sulfated GalNAc of pituitary glycoprotein hor- mones such as lutropin and thyrotropin (3-5, 16, 17); (ii) 3- 0-methyl-Galp4GalNAc and FucaPGalp3GalNAc containing N-linked oligosaccharides from hemocyanin of Lymnaea stag- nalis (6); and (iii) N-linked oligosaccharides containing sial- ylated GalNAc of Bowes melanoma tissue plasminogen acti- vator (8). Unsubstituted triantennary N-linked oligosaccharides containing terminal GalNAc are thought to be present in the glycoproteins obtained from Schistosoma mansoni (7). During the preparation of this paper, Skelton et al. (31) have reported that adrenocorticotropin synthesized by AtT-20 cells had highly branched N-linked oligosaccharides with terminal GalNAc. In the present study, we confirmed in detail the structure of a variety of highly branched N-acetyllactosamine- type carbohydrate chains terminating unsubstituted GalNAc of human urinary kallidinogenase.

Green et al. (18, 19) reported that several lectins, L-PHA, E-PHA, RCA I, and RCA I1 have distinct oligosaccharide

specificity and differentiate between structures bearing terminal Gal and GalNAc residues. The addition of GalNAc residues to oligosaccharides might increase hydrophobicity of carbohydrate moiety of glycoproteins. Indeed, the delay of elution time of oligosaccharides containing GalNAcs on reverse-phase column chromatography compared with those of the usual counterparts containing Gal were observed in the present experiments. The hydrophobicity of the N-acetyl group of GalNAc residue might affect the affinity of some lectins.

Smith et al. reported that pituitary GalNAc transferase specifically recognize the tripeptide sequence, Pro-Leu-Arg, that is found 6-9 residues on the N-terminal side of glycosy- lated asparagine on the a subunit and p subunit of human lutropin (20-22). Human urinary kallidinogenase protein, however, does not contain such a tripeptide sequence (1, 28, 29). GalNAc transferase in human kidney, the source of urinary kallidinogenase (23-25), might have some different


substrate recognizing structure from that in pituitaries. Baenziger et af. (26) and Fiete et af. (27) reported that

hepatic reticuloendothelial and Kupffer cells bind specifically S04-4GalNAc/34GlcNAc~2Mancu sequence and they proposed a major mechanism for clearance of certain sulfated glycoproteins from the blood. Although sample kallidinogenase protein was purified from fresh human urine, any sulfate group was not detected. Whether human kallidinogenase contains a sulfate group or not when it is synthesized in kidney is indeed an interesting question.

Acknowledgment-We thank Y. Wada for her excellent technical assistance.

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