Eur. J. Biochem. 267, 2105±2112 (2000) q FEBS 2000

Structural characterization of the oligosaccharide chains of humana1-microglobulin from urine and amniotic fluid

Angela Amoresano1, Lorenzo Minchiotti2, Maria E. Cosulich2, Monica Campagnoli2, Piero Pucci1,Annapaola Andolfo1, Elisabetta Gianazza3 and Monica Galliano2

1Centro Internazionale di Servizi di Spettrometria di Massa e Dipartimento di Chimica Organica e Biologica, UniversitaÁ di Napoli, Italy;2Dipartimento di Biochimica `A. Castellani', UniversitaÁ di Pavia, Italy; 3Istituto di Scienze Farmacologiche, UniversitaÁ di Milano, Italy

Human a1-microglobulin (a1-m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily,

was isolated by sequential anion-exchange chromatography and gel filtration from the urine of hemodialized

patients and from amniotic fluid collected in the week 16±18 of pregnancy. The carbohydrate chains of the

protein purified from the two sources, which are organized in two Asn-linked and one Thr-linked oligo-

saccharides, were structurally characterized using matrix-assisted laser desorption ionization and electrospray

mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and

O-glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary

structures attached in the case of urinary a1-m and only diantennary glycans in the amniotic fluid protein. The

main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary

glycans. The position of the oligosaccharide chains in a three-dimensional model of the protein, produced using

the automated Swiss-Model service, is also discussed.

Keywords: lipocalins; mass spectrometry; a1-microglobulin; oligosaccharide structure.

Human a1-microglobulin (a1-m, also known as protein HC) is alow molecular mass glycoprotein present in serum in a free,monomeric form as well as in high molecular mass complexeswith other plasma proteins [1±3]. It was originally isolated andcharacterized from the urine of patients with chronic cadmiumpoisoning [3,4] and is a clinical marker of tubular proteinuria.However, a1-m is also widely distributed in other body fluids,including amniotic fluid, from which it has recently beenpurified and spectroscopically characterized [5].

The polypeptide chain of mature a1-m, which consists of183 amino acids [6,7], contains one O-linked (Thr5) and twoN-linked (Asn17 and Asn96) glycosylation sites with attachedglycans of known composition, but unknown primary structure[8,9]. The three-dimensional structure of the protein has notbeen determined but, based on its sequence homology, a1-mhas been included in the lipocalin family. This is a group ofsmall, predominantly extracellular proteins best known for theirbinding of small hydrophobic ligands, but which have recentlybeen assigned other roles such as those of enzymes, immuno-modulators and regulators of cell homeostasis [10]. Despite

their functional diversity and low sequence homology, theoverall folding pattern common to the lipocalins is a highlyconserved b-barrel motif [10]. In this group a1-m is uniquebecause it has a major yellow-brown fluorescent chromophoreand displays extensive charge and size heterogeneity [1,3]. Theidentity of the strongly bound ligand, which shows severalfeatures different from the prosthetic groups of other lipocalins,is still unknown.

The biological role of a1-m has been investigated during thelast two decades, but so far no clear function of the protein hasbeen identified. The plasma concentration of free a1-m, whichis in the range 20±30 mg´L21, does not change significantlyduring inflammatory or neoplastic conditions. However, severalreports hint at a role for a1-m as an immunomodulator. Theprotein inhibits the chemotactic response of normal neutrophilsto endotoxin-activated serum [11], the stimulation of lympho-cytes by protein antigens [12], and the production ofinterleukin-2 by T-helper cells [13]. In addition to its inhibitoryeffects on the immune system, a1-m also exhibits mitogenicproperties [1]. It has also been proposed that most or all of theimmunosuppressive effects of a1-m could be accounted for byits oligosaccharide moieties [1]. Another lipocalin, glycodelin,has potent immunosuppressive and contraceptive activity thathas been shown to be related to its gender-specific glycosyla-tion, which regulates some key processes related to humanreproduction [14]. As these immunoregulatory properties mightbe tissue-specific, the elucidation of the structure of theglycosidic moiety and the distribution of glycoforms in a1-misolated from different tissues might shed light on the biologicalrole played by the glycans of this lipocalin. This paper reportsthe structure of the overall glycosidic moieties of a1-m purifiedfrom human urine and amniotic fluid and describes the oligo-saccharide chains present at each individual N-glycosylationand O-glycosylation site.

Correspondence to M. Galliano, Dipartimento di Biochimica

`A. Castellani', UniversitaÁ di Pavia, via Taramelli 3B, I-27100 Pavia, Italy.

Fax: 139 0382423108, Tel.: 139 0382507724, E-mail: galliano@unipv.it

Abbreviations: a-cyano, a-cyano-4-hydroxycinnamic acid; a1-m,

a1-microglobulin; DHB, 2,5-dihydroxybenzoic acid; ESMS, electrospray

mass spectrometry; Hex, hexose; HexNAc, N-acetyl hexosamine; PNGase

F, peptide N-glycosidase F; RP, reverse phase.

Enzymes: peptide N-glycosidase F (EC 3.2.2.18) from Flavobacterium

meningosepticum; neuraminidase (sialidase), acylneuraminyl hydrolase

(E.C. 3.2.1.18) from Vibrio cholerae; trypsin (EC 3.4.21.4) from bovine

pancreas.

(Received 24 November 1999, revised 9 February 2000, accepted

11 February 2000)

2106 A. Amoresano et al. (Eur. J. Biochem. 267) q FEBS 2000

M A T E R I A L S A N D M E T H O D S

Materials

Trypsin, dithiothreitol, iodoacetic acid, 2,5 dihydroxybenzoicacid (DHB) and a-cyano-4-hydroxycinnamic acid (a-cyano)were purchased from Sigma. Peptide N-glycosidase F (PNGase F)was obtained from Boehringer. Cyanogen bromide was purchasedfrom Pierce. Pre-packed PD-10 gel filtration cartridges were fromPharmacia; prepacked Sep-pak C18 cartridges were purchasedfrom Waters. All other reagents and solvents were of the highestpurity available from Baker.

Isolation of urinary a1-m

Urine of hemodialized patients (1.5 L) were saturated to80% with ammonium sulfate and the precipitate collected bycentrifugation at 10 000 g for 1 h, dissolved in 50 mm sodiumacetate, pH 5.8, and applied to a DEAE-cellulose 2.6 � 30 cmcolumn. After the unbound proteins were washed out with thesame buffer, the column was eluted with 50 mm sodiumacetate, pH 5.8, 300 mm NaCl. The eluted proteins werepooled, concentrated and fractionated by gel-filtration on a2.6 � 100 cm Sephadex G 100 column eluted with 20 mmTris/HCl, pH 8.0, 150 mm NaCl. The homogeneity of thea1-m containing fractions was checked by SDS/PAGE underreducing and nonreducing conditions in 17% gels and byN-terminal sequence analysis in a Hewlett-Packard model G1000 A sequencer (Centro Grandi Strumenti, UniversitaÁ diPavia).

Production of a monoclonal antibody specific for a1-m

A monoclonal antibody specific for the protein was producedby somatic cell fusion; lymphocytes from Balb/c mouseimmunized with purified urinary a1-m were fused withP3X63Ag8 myeloma cells.

A clone named a1 4/9 (Ig G1k) was selected: it specificallyreacts with a1-m both in ELISA and Western blot analysis. Theepitope was localized within the N-terminal region of themolecule (residues 1±41).

Isolation of amniotic a1-m

Aliquots of human amniotic fluid collected in the weeks 16±18of pregnancy were pooled (1 L) and dialyzed against 6.25 mmBistris-propane, pH 7.5, and fractionated on a 2.6 � 30 cmhigh performance Q-Sepharose column equilibrated with thesame buffer (buffer A) and connected to an AKTA purifiersystem (Pharmacia). The elution was performed with a 375 minlinear gradient from 0±100% of 6.25 mm Bistris-propane,pH 9.5, 350 mm NaCl (buffer B) and monitored at 280, 330 and440 nm. Fractions were manually collected and analyzed bySDS/PAGE under reducing and nonreducing conditions in 17%gels followed by immunoidentification by western blot. Thea1-m containing fractions were pooled, concentrated andfractionated by gel-filtration on a Superdex 75 column(Pharmacia) connected to the AKTA purifier system, equili-brated and eluted with 20 mm Tris/HCl, pH 8.0, 150 mm NaCl.The homogeneity of the protein was checked by N-terminalsequence analysis in the Hewlett-Packard model G 1000 Asequencer.

Analytical gel electrophoresis

Isoelectric focusing was performed on laboratory madepolyacrylamide gels cast on GelBond, dried and rehydrated tooriginal thickness of 0.5 mm with a solution containing 8 murea and carrier anpholytes in the pH range 3.0±10.0 [15].SDS/PAGE was performed on 17% polyacrylamide gels using aMini PROTEAN II cell (BioRad).

Immunodetection of a1-m was performed by western blottechnique: proteins were electrotransferred from SDS/PAGE toan Immun-Blot poly(vinylidene difluoride) membrane (Biorad)and incubated with anti-(a1-m) monoclonal antibody at5 mg´mL21 final concentration. Secondary antibody goatanti-(mouse IgG) Ig labeled with peroxidase (SouthernBiotechnology) were used according to the manifacturer'sinstructions (1 : 1000, v/v final concentration). Peroxidasewas revealed by enhanced chemioluminescence (AmershamPharmacia Biotech) detected by autoradiography (Hyperfilm;Amersham Pharmacia Biotech).

Chemical and enzymatic hydrolyses

Aliquots (15 mg) of the two pure proteins were desialylatedby treatment with neuraminidase from Vibrio cholerae(Boehringer) in 50 mm sodium acetate, 154 mm NaCl, 9 mmcalcium chloride at 37 8C overnight and directly applied ontothe isoelectric focusing gel.

Protein samples were reduced and alkylated with iodoaceticacid according to the procedure previously described [16].Tryptic digestion was carried out in 50 mm ammoniumbicarbonate, pH 8.5, at 37 8C overnight using an enzyme tosubstrate ratio of 1 : 50. CNBr hydrolysis was accomplishedwith a 15-fold excess of CNBr (mass/mass) in 70% trifluoro-acetic acid overnight at room temperature in the dark. Theprotein sample was then diluted 10-fold with cold water,evaporated in a Speed-Vac centrifuge (Savant) and lyophilized.CNBr and tryptic peptide mixtures were fractionated by RP(reverse phase)-HPLC on a Vydac C18 column (25 � 0.46 cm,5 mm) using 0.1% trifluoroacetic acid (solvent A) and 0.07%trifluoroacetic acid in 95% acetonitrile (solvent B) by means ofa linear gradient from 5±65% solvent B over 60 min.

Deglycosylation of the various glycopeptides was performedby treatment with PNGase F in 50 mm ammonium bicarbonate,pH 8.5, overnight at 37 8C using 0.1 enzyme unit per 0.5 mg ofprotein.

Isolation and derivatization of oligosaccharide chains

Total N-linked glycans released by PNGase F were separatedfrom peptides by reverse phase chromatography on prepackedSep-pak cartridges (Waters) equilibrated with 5% acetic acid.The oligosaccharides were eluted in the aqueous phase andfreeze-dried whereas peptides were retained on the cartridgeand eluted with 5% acetic acid in 40% propanol. Glycans werepermethylated using the sodium hydroxide procedure toincrease the sensitivity of the MALDI MS analyses [17].Briefly, 0.5±1 mL of dimethyl sulfoxide/NaOH slurry (pre-pared by grinding five pellets of sodium hydroxide withapproximately 3 mL of dry dimethyl sulfoxide) was added tothe oligosaccharide sample followed by about 0.5 mL ofmethyl iodide. The mixture was shaken for 10 min at roomtemperature and then quenched with 1 mL of water. Theproducts were extracted into chloroform, which was washedseveral times with water. The chloroform layer was dried under

q FEBS 2000 Glycans of human a1-microglobulin (Eur. J. Biochem. 267) 2107

a stream of nitrogen and the residue was dissolved in methanolfor MALDI MS analysis.

Mass spectrometry

MALDI mass spectra were recorded in the positive-ion modeusing a Voyager DE MALDI-TOF mass spectrometer (Perkin-Elmer) equipped with the delayed extraction. The MALDImatrices were prepared by dissolving 10 mg of DHB in 1 mLmethanol or 10 mg a-cyano in 1 mL of acetonitrile/0.2%trifluoroacetic acid (70 : 30, v/v). Typically, 1 mL of matrixwas applied to the metallic sample plate and 1 mL of analytewas then added. Mass calibration was performed with the MH1

ion of insulin set at m/z 5734.6 and an a-cyano peak at m/z379.6 or a known peptide ion at m/z 1209.7. Raw data wereanalysed using a computer software provided by the manu-facturers and are reported as average masses.

Electrospray mass spectra were obtained with an API 100single quadrupole instrument (Perkin Elmer) or a BIO-Q triple

quadrupole mass spectrometer (Micromass). Peptide sampleswere dissolved in 1% acetic acid in 50% acetronitrile andinjected into the ion source at a flow rate of 6 mL´min21.Spectra were acquired and elaborated using the softwareprovided by manufacturers. Calibration of the mass spectro-meter was achieved by means of a separate injection ofpolyethylene glycol or myoglobin. All masses reported areaverages.

Tertiary structure

The tertiary structure of a1-m has been deduced using theautomated Swiss-Model service, which extrapolates a modelfor a target sequence from the known three-dimensionalstructure of related family members (template) [18±20]. TheNMR structure of human neutrophil gelatinase associatedlipocalin [21], which shows 25.7% sequence identity with a1-m(entry 1ngla in the Brookhaven Protein Data Bank) was used asa template.

Fig. 1. Q-Sepharose anion-exchange

chromatography of the amniotic fluid. Human

amniotic fluid (300 mL) was dialyzed against

6.25 mm Bistris-propane, pH 7.5, and injected

on a 2.6 � 30 cm high performance

Q-Sepharose column equilibrated with the same

buffer (buffer A) connected to an AKTA purifier

system (Pharmacia). The elution was performed

at a flow rate of 5 mL´min21 with a 375 min

linear gradient from 0±100% of 6.25 mm

Bistris-propane, pH 9.5, 350 mm NaCl (buffer

B). The dashed line denotes the absorbance at

330 nm: 0.1 full scale. Peak numbers correspond

to the following proteins: 1, b2-microglobulin;

2, transferrin; 3, a1-m, a1-antitrypsin; 4,

albumin. The eluate was checked by

immunoblotting and the a1-m containing

fractions were pooled as indicated by the

horizontal bar. The other proteins were

identified by N-terminal sequence analysis after

further purification by gel filtration.

Table 1. Oligosaccharide structures observed in the MALDI spectra of the permethylated glycans released from individual N-glycosylation sites in

urinary and amniotic a1-m.

MNa1

Glycosylation site Urinary Amniotic Structure

Asn17 2070�.6 2070�.5 Diantennary

2227.6 Monosialylated diantennary (truncated)

2432.3 2434�.6 Monosialylated diantennary

2791.7 2791�.4 Disialylated diantennary

Asn96 2070�.6 2070�.5 Diantennary

2227�.6 Monosialylated diantennary (truncated)

2244.3 Fucosylated diantennary

2432.3 2434�.6 Monosialylated diantennary

2605.1 2605�.3 Fucosylated monosialylated diantennary

2791.7 2791�.4 Disialylated diantennary

2966.5 2964�.9 Fucosylated disialylated diantennary

3603.9 Trisialylated triantennary

3777.4 Fucosylated trisialylated triantennary

2108 A. Amoresano et al. (Eur. J. Biochem. 267) q FEBS 2000

R E S U L T S

Isolation and electrophoretic analysis of urinary andamniotic fluid a1-m

The final recovery of a1-m isolated from the urine ofhemodyalized patients (1.5 L) in a two-step procedure was380 mg. The yellow-brown protein gave a single diffuse bandof about 30 kDa on SDS/PAGE but, when analyzed byisoelectric focusing, showed both discrete and broad bandsdistributed between pH 3.8±5.0. Edman degradation of the first10 N-terminal residues revealed a single amino acid sequence:Gly-Pro-Val-Pro-Xaa-Pro-Pro-Asp-Asn-Ile, identical to thatreported in the literature [6,7]. An aliquot (5 mg) of the urinaryprotein was used as antigen to produce a monoclonal antibodythat was then used to probe for a1-m in the amniotic fluid.

The amniotic fluid was fractioned by anion-exchangechromatography on Q-Sepharose (Fig. 1). The a1-m containingfractions immunodetected by mAb 4/9 were collected andfurther purified by gel filtration on a Superdex 75 column, witha recovery of 5 mg of protein per liter of starting material. Thelight yellow protein behaved as urinary a1-m in SDS/PAGE andon isoelectric focusing showed a similar charge heterogeneity,with a diffuse band in the pH range 4.0±4.8. The identity of theprotein was confirmed by N-terminal sequence analysis.

Characterization of the glycans from urinary a1-m

The structure of the overall glycosidic moiety of urinary a1-mwas investigated by direct mass analysis of the permethylatedoligosaccharide mixtures. The glycoprotein was reduced andcarboxymethylated under denaturing conditions using iodo-acetic acid and digested with CNBr. The intact N-linkedoligosaccharides were released from the peptide backbone byPNGaseF treatment of the CNBr digest. The glycan mixturewas separated from the peptides by a Sep-pack reverse phasechromatographic step, permethylated and analysed by MALDIMS. The MALDI spectrum of the corresponding oligosaccha-ride mixture is shown in Fig. 2A. The different glycan struc-tures were identified by their mass values on the basis of theknown biosynthetic pathway of N-linked glycan structuresand are reported in Table 1. The N-linked glycans from urinarya1-m constitute a largely heterogeneous mixture of complextype structures. The most abundant species consist essentiallyof disialylated diantennary structures. Minor components cor-responding to fucosylated and non fucosylated diantennary andtriantennary oligosaccharides carrying a different number ofsialic acid molecules were also detected as indicated in Table 1.

Since a1-m contains an O-linked and two N-glycosylationsites, a detailed study aimed at defining the site-specificity ofglycosylation was also carried out. The protein was treated withCNBr and the peptides were separated by HPLC as shown inFig. 3 and directly analysed by electrospray mass spectrometry(ESMS). A perfect agreement was observed between themeasured and the theoretical mass values for all the peptideswith the exception of fractions 3, 6 and 7 (Table 2). The ESMSspectrum of fraction 3 indicates the presence of essentiallythree components with the major species displayingmolecular masses of 6573.9 ^ 0.9 Da, 6283.6 ^ 0.6 Da and5991.7 ^ 0.9 Da. On the basis of their mass values, thesecomponents were identified as peptide residues 63±99,containing Asn96, carrying disialylated, monosialylated andnonsialylated diantennary complex structures, respectively.Other minor satellite peaks whose mass values were measured

Fig. 2. MALDI mass spectra of permethylated oligosaccharides released

from urinary (A) and amniotic (B) a1-m. Samples were dissolved in

methanol and mixed with the matrix prepared by dissolving 10 mg of DHB

in 1 mL methanol. Spectra were acquired at 25 kV acceleration voltage by

summing 250 laser shots. All mass signals correspond to the sodium

adducts (MNa1) of the oligosaccharide structures.

q FEBS 2000 Glycans of human a1-microglobulin (Eur. J. Biochem. 267) 2109

as 6718.8 ^ 0.2 Da and 6427.1 ^ 0.1 Da, corresponding tothe fucosylated forms of the glycopeptide, were also present.Fraction 6 represents the fragment 45±99, which originatedfrom an uncompleted cleavage at Met62 and is modified bymonosialylated and disialylated diantennary structures.

The ESMS spectrum of fraction 7 is shown in Fig. 4A; thepresence of four molecular species can be observed. On thebasis of their mass values, these components were tentativelyidentified as the N-terminal peptide 1±41, differently modifiedby various glycan structures. As this peptide contains both theO-glycosylation and N-glycosylation sites at Thr5 and Asn17,the structure of the individual glycoforms linked to eachposition could not be defined at this stage.

Fractions 3 and 7 were then deglycosylated by PNGase Ftreatment, which specifically releases N-linked glycans leavingunaffected the O-linked structures. The oligosaccharides wereseparated from the peptidic material by HPLC and permethy-lated before MALDI MS analysis. The deglycosylated fraction7 was re-examined by ESMS and it generated the spectrumshown in Fig. 4B. Two species were detected in thespectrum displaying mass values of 5358.6 ^ 0.6 Da and5067.9 ^ 0.3 Da, respectively. These mass values were inter-preted as arising from the peptide 1±41 still carrying O-linkedglycans of the HexHexNAc structure with one or no sialicacid molecule. The mass difference observed before andafter deglycosylation suggested that Asn17 was modified

Fig. 3. HPLC separation of CNBr peptides

from reduced urinary a1-m. Peptides were

resolved on a Vydac C18 column (25 � 0.46 cm,

5 mm) using 0.1% trifluoroacetic acid (solvent

A) and 0.07% trifluoroacetic acid in 95%

acetonitrile (solvent B). A linear gradient from

5±65% solvent B over 60 min was employed.

The individual fractions are indicated by

numbers.

Table 2. ESMS analysis of the reduced and carboxymethylated urinary a1-m digested with cyanogen bromide and fractionated by HPLC. Peptides

were identified within the protein sequence on the basis of their molecular mass and the CNBr specificity. All fragments with the exception of the C-terminal

peptides were recovered in the lactone form.

Molecular mass

No. Expected Measured Peptide Glycosylation site

1 1670�.8 1670.3 �^ 0.5 164±178

2 1783�.9 1784.2 �^ 0.6 164±179

3 5989�.2 5991.7 �^ 0.3 63±99 1 diantennary Asn96

6280.5 6283.6 �^ 0.6 63±99 1 diantennarySia Asn96

6571.8 6573.9 �^ 0.9 63±99 1 diantennarySia2 Asn96

4 2263�.6 2263.4 �^ 0.4 164±183

5 1759�.9 1759.7 �^ 0.4 45±62

6 8040�.4 8042.4 �^ 0.8 45±99 1 diantennarySia Asn96

8331.7 8333.0 �^ 0.9 45±99 1 diantennarySia2 Asn96

7 6689�.1 6690.9 �^ 0.7 1±41 1 glycans Asn17,Thr5

6980.4 6983.8 �^ 0.8 1±41 1 glycans Asn17,Thr5

7271.7 7273.9 �^ 0.9 1±41 1 glycans Asn17,Thr5

7563.0 7565.5 �^ 0.7 1±41 1 glycans Asn17,Thr5

8 7309�.3 7312.1 �^ 0.7 100±163

9 7309�.3 7312.1 �^ 0.9 100±163

2110 A. Amoresano et al. (Eur. J. Biochem. 267) q FEBS 2000

essentially by diantennary structures with varying degrees ofsialylation.

These results were essentially confirmed by the MALDIanalysis of the glycan moieties released by PNGaseF treatment(Table 1). Mass spectral investigation of the oligosaccharidesfrom Asn17 showed the presence of diantennary structurescarrying a different number of sialic acid residues. The glycansreleased from Asn96 displayed a slightly more heterogeneousmixture of oligosaccharides consisting of diantennary andtriantennary structures. These data indicate that a difference inthe site-specificity of glycosylation exists in the urinary proteinwith Asn17 bearing essentially diantennary glycans and thetriantennary oligosaccharides being only attached to Asn96.

Characterization of the glycans from amniotic fluid a1-m

The structural characterization of the N-linked glycans releasedfrom amniotic fluid a1-m was accomplished essentially follow-ing the procedure described above. The glycoprotein wasreduced, alkylated and digested with trypsin. The oligosaccha-rides were released by PNGase F treatment, permethylated anddirectly analysed by MALDI MS. The corresponding MALDIspectra (Fig. 2B) showed the presence of two series of threerelated signals at m/z 2791.4, 2434.6, 2070.5 and 2964.9,2605.3, 2244.3, respectively. The three signals within eachseries refer to diantennary structures carrying two, one and nosialic acids on the antennae. The mass difference between thetwo series of related signals was due to the presence of a fucoseresidue. These results demonstrate that the glycans N-linked toamniotic fluid a1-m belong to the complex type oligosaccha-rides and consist only of diantennary structures, showing anarrower distribution of glycoforms with respect to the urinarya1-m.

The site-specificity of the N-glycosylation in the amnioticprotein was investigated following essentially the proceduredescribed above using trypsin as proteolytic agent. Both thedirect analysis of the glycopeptides by ESMS following HPLCfractionation of the tryptic digest and the MALDI MS spectraof the glycans released from each glycosylation site by PNGaseF provided identical results. Both Asn17 and Asn96 displayedthe presence of diantennary complex type oligosaccharideswith varying degrees of sialylation with fucosylation onlyoccurring at the glycans attached to Asn96. The ESMSspectra of the N-terminal peptide 1±20 led us to check theO-glycosylation site occurring at Thr5. Again only glycosidicstructures of the HexHexNAc type with varying degrees ofsialylation were detected. However, O-glycosylation in theamniotic fluid a1-m was only partial as indicated by theoccurrence of the unmodified N-terminal peptide 1±20(2224.3 ^ 0.1 Da).

D I S C U S S I O N

The glycosidic moieties of a1-m isolated from human urine andamniotic fluid were structurally characterized in detail usingmass spectrometric methodologies. The carbohydrate units ofthe proteins were determined by analysis of the glycopeptidesformed after CNBr or tryptic cleavage. On the basis of itsprimary structure [6], a1-m has two consensus sequences,Asn-Xaa-Thr/Ser, for N-glycosylation (Asn17 and Asn96) andone potential linkage site (Thr5) for O-glycans [22].

Though there is no total agreement as to the exact amino acidsequence that defines an O-glycosylation site, the presence of acluster of Pro residues adjacent to a Thr is known to favoroligosaccharide addition [23]. The O-linked structures attached

Fig. 4. Transformed ESMS spectrum of (A) fraction 7 and (B)

deglycosylated fraction 7. Spectra were obtained by using a cone voltage

of 40 V and an acceleration voltage of 3.8 kV. Samples were introduced into

the ion source at 6 mL´min21. Individual species are indicated by capital

letters; the molecular masses of the various components are reported.

q FEBS 2000 Glycans of human a1-microglobulin (Eur. J. Biochem. 267) 2111

to Thr5 are different HexHexNAc glycans, with the occurenceof only partial O-glycosylation in the amniotic protein.

Qualitative and quantitative differences could be detected inthe relative amount of oligosaccharide components attached tothe N-glycosylation sites of urinary and amniotic glycoproteins.Differences, in fact, exist in the relative amount of thesecomponents as well as in the presence of different structureswithin the two proteins. Urinary a1-m showed a higher hetero-geneity of the glycosidic moiety as well as the occurrence ofhigher branched structures with respect to the amniotic fluidglycoprotein. Moreover, as detected by site-specificity experi-ments, diantennary and triantennary structures represent theglycoforms occurring at Asn96 of urinary a1-m whereasdiantennary glycans constitute the only structures occurring atthe same site of the amniotic protein. A lower degree ofheterogeneity has been found in the carbohydrate attached toAsn17, in which monosialylated and disialylated diantennaryglycans represent the main stuctures identified in both proteins.Among the a1-m homologs, the consensus sequence of theposition corresponding to Asn17 of the human protein is poorlyconserved. Based on available cDNA or amino acid sequences,this site is absent in other mammalian species, including gerbil,rat, cow and pig, as well as in salmon, frog, and plaice, whereasit is conserved only in mouse and hamster a1-m [24]. Theexistence of such species-specific variations in a conservedprotein suggests that the oligosaccharide chain at this siteprobably does not play crucial roles in the basic functionof a1-m. On the contrary, the consensus sequence forN-glycosylation involving Asn96 of the human protein isconserved among all known mammalian a1-m. It is interestingto note that this sequence, Asn-Ile-Thr, occurs within theglycopeptides isolated from the human and pig proteins thatinhibit antigen-specific stimulation of human peripheral bloodleucocytes [25].

Previous data on the carbohydrate composition of a1-mshowed that the sugars of the urinary and plasma proteins arenearly identical and suggested the presence of an unbranchedO-GalNAc-GalNAc-Neu chain at Thr5, of triantennary or tetra-antennary structures at Asn17, and of diantennary glycans at

Asn96, respectively [9]. Our results show a higher degree ofheterogeneity of both the O-linked and N-linked oligosaccha-ride chains and indicate that the contribution of the sugar to themolecular mass of the protein is about 15%, a result somewhatlower than the previously reported 23% [9], but in agreementwith the value suggested by EkstroÈm et al. [8].

The peculiar extensive charge heterogeneity of this proteinhas been attributed mainly to the variations in structure and/oramount of its chromophore [5], although the existence ofmolecules of a1-m with and without the C-terminal basictetrapeptide, due to varying degrees of proteolytic breakdown,has also been described [6]. In the present work we haveidentified in the urinary protein three different polypeptidechains of 178, 179 and 183 amino acids, respectively, becauseof the presence of a previously unreported minor form lackingthe five C-terminal residues (Table 2 and Fig. 3). Moreover, theidentification of a highly heterogeneous mixture of differentlybranched glycans suggests that differences in carbohydrates canmake a contribution to the broad isoelectric pattern of a1-m.

The expression of specific types of glycosylation on the sameprotein isolated from two different biological fluids suggeststhat these structures may have diverse roles at different times ofdevelopment. Urine of patients with renal failure represents atypical source for the isolation of adult a1-m, which is presentin plasma in quite low concentration, representing only0.03±0.04% of the total protein content. On the other hand, theprotein is relatively more abundant in the amniotic fluid, inwhich the fetal urinary excretion is the major determinantof a1-m content, especially during the second trimester ofgestation [26,27]. In this period its level is 28.4±34.5 mg´L21,which represents about 0.8% of the total protein amount, andthereafter decreases progressively to 14.1 mg´L21 near term[26], as a consequence of an increasing tubular reabsorptioncapacity.

Several reports indicate that oligosaccharides associated withsoluble glycoconjugates found in the human amniotic fluidparticipate in the mechanism underlying the protection of thehuman fetus from the maternal immune response [28]. Therelatively few examples in which such glycan structures havebeen characterized and their biological functions clearlydefined includes the selectin family [14,29]. Both the O-linkedand the N-linked glycans of a1-m are commonly occuringcarbohydrate chains. Therefore, a straightforward relationshipwith the proposed immunomodulatory role of the proteincannot be deduced. However, a proper spacing on the linearpolypeptide chain or in the proper three-dimensional contextmight allow the generation of uncommon recognition markersusing common oligosaccharides [30]. In this light we haveexamined the tertiary structure of a1-m extrapolated from theknown three-dimensional structure of the related humanneutrophil gelatinase associated lipocalin [18±21]. In thismodel (Fig. 5) the N-linked glycosylation sites occur in twoexposed loops, while the O-linked site is located in theN-terminal stretch which, in the template as well as in themodel we have produced, is not structured. In the modelthe glycans of a1-m are on the same plane on the molecularsurface at the three vertices of a triangle and are an average of24 AÊ from each other. As protein±protein interactions usuallyinvolve small surface areas, it seems unlikely that they may allplay a role in a specific molecular recognition.

The highly hydrophilic oligosaccharide chains are allowedseveral conformations and are highly flexible in the solventenvironment. Therefore, the suggestion that they are localizedon the same side of the molecule and the finding of a differentglycosylation patterns at different times of development

Fig. 5. The Swiss-Model deduced tertiary structure of a1-m. The model

is generated by the automated Swiss-Model service. The side chains of the

three glycosylation sites are indicated.

2112 A. Amoresano et al. (Eur. J. Biochem. 267) q FEBS 2000

indicate that the glycans of a1-m might provide modulation ofbiological processes involving the protein.

A C K N O W L E D G E M E N T S

We gratefully acknowledge the technical assistance of Antonio Mortara and

Marco Bellaviti. We wish to extend our gratitude to Drs Gianlodovico Melzi

d'Eril (UniversitaÁ dell'Insubria, Varese), Claudio Bonfanti (Ospedale S.

Anna, Como) and Francesco Poggio (Fondazione S. Maugeri, Pavia) for

supplying the biological fluids. We thank Dr Alessandra Cobianchi (Centro

Grandi Strumenti, UniversitaÁ di Pavia) for sequence analysis, Dr Hugo L.

Monaco for his critical reading of the manuscript and Dr Massimiliano

Perduca for help with the preparation of the three-dimensional model

figure. This work was supported by PRIN (Progetto di ricerca di interesse

nazionale) grant `Structural studies on hydrophobic molecule-binding

proteins' from the Ministero della UniversitaÁ e della Ricerca Scientifica e

Tecnologica (Rome, Italy).

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