Human granulocyte/pollen binding protein Recognition and identification as transferrin

Human granulocyte/pollen-binding protein.

Recognition and identification as transferrin.

S P Sass-Kuhn, … , O Cromwell, A B Kay

J Clin Invest.

1984;

73(1)

:202-210.

https://doi.org/10.1172/JCI111192

.

Normal human serum was found to contain a heat-stable protein which promoted the

binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]).

GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually

all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a

molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1.

By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis,

the protein was identified as transferrin. Monospecific antisera raised against either GPBP

or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin

gave lines of identity with these two antisera. The apparent heterogeneity in the molecular

size and charge of GPBP observed during progressive purification was minimal when

GPBP was saturated with ferric ions before the separation procedures. These experiments

indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin

which appears to be unrelated to iron transport and raises the possibility that transferrin

might have a physiological role in the removal of certain organic matter.

Research Article

A

bstract. Normal human serum was

found

to

contain

a

heat-stable protein which

promoted the binding

of granulocytes

to

timothy

grass pollen

(granulocyte/pol-len-binding

protein

[GPBP]).

GPBP was purified by gel

filtration, anion

exchange, and affinity

chromatography.

Virtually

all

of the

granulocyte/pollen-binding

activity

was

associated

with a

_#-

1-protein

having a molecular mass

of

-

_77,000

_D

_and

_an

_{isoelectric point of}

_{between 5.5}

and

6.1.

By

immunoelectrophoresis

and

sodium dodecyl

sulfate-polyacrylamide

gel

electrophoresis,

the

protein

was

identified

as

transferrin.

Monospecific antisera raised

against

either GPBP

or

transferrin

removed biological

activity from

GPBP

preparations,

and

GPBP and

trans-ferrin

gave

lines of identity with

these two antisera. The

apparent

heterogeneity in

the molecular size and charge

of GPBP observed during progressive purification

was

minimal

when GPBP was saturated

with ferric

ions before

the

separation

procedures.

These

experiments

indicate

that

granulocyte/pollen binding is

a

hitherto

unrecognized

property

of

transferrin

which

appears

to

be

unrelated

to

iron

transport

and raises the

possibility

that transferrin

might

have

a

physiological

role in

the

removal of certain

organic

matter.

Introduction

During the

course of

experiments designed

todetermine whether

various allergens activate the alternative

pathway

of

complement,

we made the

unexpected

observation thatnormal humanserum,

either

heated or

unheated, promoted firm, prolonged binding

Address allcorrespondencetoDr.Kay.

Received

for publication

20April1983 and in

revisedform

19

Sep-tember1983.

J.Clin.Invest.

©The American

Society

forClinicalInvestigation,Inc.

0021-9738/84/01/0202/09 $1.00 Volume73, January 1984,202-210

Human

Granulocyte/Pollen-binding Protein

Recognition and

Identification

as

Transferrin

SuzanneP.Sass-Kuhn, R. Moqbel, Judith A. Mackay,

0. Cromwell, and A. B. Kay

Department ofAllergyand ClinicalImmunology, Cardiothoracic

Institute, BromptonHospital,London, United Kingdom

of

granulocyte to

timothy

grass pollen

(TGP).'

We had

under-taken these

studies in

the

expectation

that sera from certain

susceptible, i.e., "atopic",

individuals might facilitate adherence

of neutrophils

and/or eosinophils to allergens via IgG or IgE orthat normalserum might produce binding through alternative

pathway

complement activation as was previously shown for

helminthic

larvae (1, 2). The finding that heated serum from a number

of

healthy normal

individuals

promoted granulocyte

binding

(irrespective of whether

they were skin

[prick]

test

pos-itive

or

negative

to the TGP extract) made it unlikely that either

IgG, IgE,

or

complement

were

involved. Accordingly,

we

pro-gressively

purified this

granulocyte/pollen-binding

protein

(GPBP) and found it

tobe

identical

to serum

transferrin.

Methods

Materials

Materials were obtained as follows. TGP (Phleum pratense) (a gift from

Dr.DavidMoran, Beecham Pharmaceuticals, Epsom, England);

trans-ferrin, IgG, vitaminB12, human serum albumin, ferric chloride (Sigma,

Poole, England); lactoferrinandantilactoferrin(agift from Dr. A. Segal,

UniversityCollege Hospital, London); antitransferrin (Dako,

Copen-hagen, Denmark); anti-whole normal human serum (Dako);

anti-f3-2-glycoprotein 1, anti-Factor B, antiplasminogen (Behring, Hounslow,

England); fibrinogen, thrombin, plasmin, aprotinin(agift from Dr. P.

Gaffney,National Institute of Biological Sciences,Hampstead,London). Theplasmin and thrombinwere World HealthOrganization International Referencepreparations and fibrinogenwas aKabi GradeLpreparation.

Sephadex G-200, Sephadex G-75, Blue Sepharose CL-6B, DEAE-Se-phacel,protein A-SepharoseCL-4B,CNBr-activated Sepharose4B,

lysine-Sepharose 4B,Blue Dextran2000,andhigh-molecularmarkersfor

so-diumdodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE)

(Pharmacia,Uppsala, Sweden);Iscove'smedium free of bovineserum

albumin, human transferrin, and soya bean lecithin, pH 7.35 (Flow

Laboratories, Irvine, Scotland); penicillin and streptomycin (Glaxo, Greenford,England).

Methods

LEUKOCYTE POLLEN ADHERENCE ASSAY

Leukocyte-rich plasma

wasobtained by sedimentation with 20% Dextran inthepresence of

1. Abbreviations usedinthis

paper: CIE,

crossed

immunoelectrophoresis;

GPBP,

granulocyte/pollen-binding

protein;

SDS-PAGE,sodiumdodecyl

preservative-free heparin (1 U/ml). Theleukocyte-rich plasmawasapplied

to adensity gradient of9% Ficoll solution and sodium diacrizoate (d

1.140) in theproportion2.4:1.Aftercentrifugationat200gfor 30 min

at20'C, thecell-richpelletwasobtained afterlysisof the erythrocytes

with 0.83% ammoniumchloride.Granulocyteswerewashedtwice with

Iscove'smediumandresuspendedto 2 X 106/ml in thesamemedium. TGPgrainsweresuspended in Iscove's mediumtogive - 1,000pollen grains/ml.Equalvolumes (50td) of leukocytes, pollen grains, andnormal human serum, orvariouschromatographicfractionsasdescribed in the

text, were mixed together in flat-bottomed microtiter plates (Nunc/ Gibco, Paisley, Scotland) and incubated for 18 hat370C inanatmosphere of 95% air and 5% Co2.Inallexperiments,the Iscove's contained 100

,gg/ml

penicillin and 100

Ag/ml

streptomycin.The number of pollens bearing adherence cells and the degree ofadherencewasestimated by eitherbright fieldordirect interferencecontrastmicroscopy(Nomarski Optics). In someexperiments, visualization of adherent leukocytesto

pollengrains wasachieved bytheaddition of 0.1%aqueoustoluidine blue. The percentage of pollen grains havingfouror more adherent granulocyteswereassessed byexamining50pollen grains.Thedegree of adherence wasrecorded accordingtothe following arbitraryscale.

(±) represents 4-7cells adherenttopollensurface; (+)representspollen

surface completely surrounded with onelayerofadherent cells;(++)

representspollensurface covered with 2-3layersof adherentcells; (+++)

represents pollen surface covered with 3-4 layers of adherent cells;

(++++) representspollen surface coveredwith morethanfourlayers

of adherent cells.Thetests wereperformedinduplicateand the variation

betweenpair intermsofpercentadherencewasusually<5%. Accuracy

was notimprovedbyincreasing the volume of the reaction mixtureto

enable >50 pollen grainstobe counted. Theratio of2,000leukocytes

to 1 pollengrainwas optimal for visualization of the degree and the

percentadherence. In mostexperiments, the resultswererecordedby

twoindependentobservers. Observer'svariationwas <10%.

Chromatography

GEL FILTRATION 3 ml of human serum were applied to an 80

X2.5-cmcolumnpreviouslyequilibrated withphosphate-bufferedsaline

(PBS)(0.01 MNaH2PO42H20,0.037MNa2HPO42H20,and0.1 M NaCl, pH 7.35). Chromatography was undertaken at

40C

withaflow

rateof20 ml/h and 2-mlfractionswere collected. Alternate fractions

were testedfor granulocyte/pollen adherence.The column was equili-bratedwith molecular weight markers (BlueDextran [2,000,000], IgG [150,000],human serum albumin [67,000], and vitamin B12 [1,350]).

Themajor peak ofactivity waspooled as indicated, dialyzedagainst

distilledwater, andIyophilized.The material wasresuspendedin PBS toavolumeof2 ml andappliedto acolumnofSephadex G-75 (80

X1.7 cm). The Sephadex G-75chromatographywasperformedin PBS at4°C withaflow rateof20 ml/h. 4-ml fractionswere collected and alternatefractionstestedfor granulocyte/pollenadherence as indicated.

Molecular weightmarkers were Blue Dextran, human serum albumin, and vitamin B12 (asfor G-200 chromatography). The fractions were

pooledasindicated,dialyzed againstdistilled water, andlyophilized.

AFFINITY. Blue Sepharose. Thelyophilized material prepared by

G-200 and G-75chromatography(Fig. 2) was resuspended in 4 ml of 0.05 MTris/HCI,pH 7.0, plus0.1MKCI,andapplied to a Blue Sepharose

CL-6B column previouslyequilibratedwiththe same buffer. The flow rate was adjusted to 60ml/hand2-ml fractions were collected. The columnsizewas 10X 1.25cm.After elution of the first major protein

peak,0.05 MTris/HCl,pH7, plus 1.5 MKC1,wasapplied as indicated. The fractionswere dialyzed for 18 h at 4°C against PBS and

50-il

samples tested forgranulocyte/protein-binding activity. Samples

con-taining activitywerepooledasindicated, dialyzed againstdistilled water for 18h,andIyophilized.

ProteinA-SepharoseCL-4B. Material obtained from BlueSepharose

was reconstituted in4mlof 0.1 M phosphatebuffer, pH 7.0,andapplied

tothe proteinAcolumn(13X 1 cm) previously equilibratedwith the samebuffer.Theexperimentwasperformedat220Cand the flow rate

was adjusted to40 ml/h; 2-ml fractions werecollected. 1 Maceticacid

wasappliedtothe columnafter themajor peakofproteinasindicated. Alternatefractionsweretestedfor GPBPactivityfollowing dialysis against PBSat4VC for 18h and the active fractions werepooled, dialyzed,and

Iyophilizedasindicated.

ANION EXCHANGE Acolumn ofDEAE-Sephacel (70X4cm)was

equilibratedwith 0.02 M sodiumphosphate buffer,0.06 MNaCi, pH

7.8. 100 ml of normal humanserum wasappliedandtheexperiment

wasperformedat4VCbyusingaflowrateof 30 ml/h. 10-mlfractions were collected.Afterthe emergence of the firstprotein peak,a linear saltgradientof 600ml, upto0.5 MNaCl,wasapplied.Alternate fractions

were assayed for GPBPactivity following dialysis againstPBS at

4VC

for 18 h.

SDS-PAGE

The discontinuousTris-glycinebuffersystemof Laemmli(3)was used for I-mm slabgelsby using12.5%acrylamide.A 3%acrylamide stacking gelwasadded toimprove bandingofproteins.Thesamples, previously lyophilized, were analyzed either in the presence orabsence of

mer-captoethanol, i.e.,3%sodiumdodecylsulfate with orwithout 5%

mer-captoethanol.Thesampleswere heated for 10minat60'Cand colored

with0.01% bromophenolblue. To each gel,40

Ag

ofproteincontained in20

AI

was added. Thesampleswereelectrophoresed at 18mA until theproteinband reached the lower 12.5%gel,atwhichtime the current wasincreasedto 30mAforanother 5 h(approximately). After

electro-phoresis,the gels werefixed for45min in 25%isopropanol/10%acetic acidand stainedovernightin0.1% CoomassieblueR250.Thegelswere destained in 8% acetic acid. Thehighmolecularweight proteinstandard

wassupplied byakit from Pharmacia Fine Chemicals(Piscataway, NJ).

Isoelectric

focusing

Thiswasperformed using LKBAmpholine RPAG plates, pH 4-6.5 (LKBInstrumentsLtd., SouthCroydon, England). 20

_Al

ofproteinwere

focused. ApH gradientwasdetermined byplacingapH electrodeat 1-cm intervals across thegelatthe conclusion oftheexperiment. The plates were stained for protein with 0.1% Coomassie blue R250. In preliminary experiments, a range of pH 3.5-9.5 was employed.Routinely,

apH range 4.0-6.5 was used.

Crossed immunoelectrophoresis (CIE)

CIEwasperformed bythe methodofLowenstein (4)withthefollowing modifications. The plates were prepared by using 8 ml of multone 1%

agarose in 25%veronalbuffer, pH 8.0, with an 8 X 8-cm glassslide. Normalserum,withorwithoutGPBP, waselectrophoresedat 5 V/cm

for 80 min in the first dimensionand at 1.4 V/cm for 20 h in the second

dimension.The 5X3.5-cm "window" contained appropriatedilutions of anti-normalhuman serum. The plates were washed for 25 h in 0.9%

NaCI,dried under filter paper, and stained with 0.5% Coomassie blue R250.

Immunoelectrophoresis

The immunoelectrophoresis was performed by the method described

byLowenstein (4) with the following modifications. 8 ml ofmultone

8-cm glassslide. The antigen was electrophoresedat5 V/cmfor 80 min. A trough was cut (5 X0.1 cm) and antiserum added. The plate was left overnight in ahumidity chamber to diffuse before washing. The plates were stained with 0.5% Coomassie blue R250.

Single radial

immunodiffusion

These wereperformed in 1% agarose, asfor CIE, or with commercial "LM-Partigen" plates (Behring, Hounslow, England).

Immunoadsorption with anti-GPBP and antitransferrin

Antisera against GPBP which gave a single line on SDS-PAGE was raised in New Zealand white rabbits according to the following schedule. Rabbits received 650ug of protein in complete Freund's adjuvant on day 1 bymultiple subcutaneous injections. 5wklater, therabbits were boosted with 150uigGPBP insaline in incomplete Freund's adjuvant intramuscularly. This procedure was repeated with 70jigGPBP at three weekly intervals for 24 wk. The IgG fraction of the rabbit antisera was prepared by ammonium sulphate precipitation and DEAE-Sephadex chromatography as follows. Saturatedammonium sulphate (30 ml) in 0.02M Tris HC1 containing 1 mMEDTA, pH 8.0, was slowly added

to50mlof the rabbit antisera and gentlymixed at room temperature for 30 min. After centrifugationat4000 gfor 45 minat4VC, the pellet was redissolved in 50 ml of 0.02 MTris HCI, 1 mMEDTA, pH 8.0. This process was repeated until thefinal precipitation was white, i.e., macroscopically free of other serum proteins. This precipitation was

redissolved in 2 ml 0.02 Msodium phosphatebuffer, pH 7.8, and dialyzed against this buffer with several changesover18 h. Thepartiallypurified

IgG was thenapplied to a DEAE-Sephacel column byusingthesame

conditions as described above, and the firstprotein peak waspooled

anddialyzed for 48 hagainstseveralchanges of distilledwaterbefore lyophilization. ThelyophilizedmaterialwascoupledtoCNBr-activated Sepharose after thefollowing procedure.28 mg of the

partially

purified

rabbit antiserawereaddedto 1.0 g of CNBr-activated Sepharose 4B.

TheSepharose waspreviously washed in 1 mM HCl andresuspended

in 0.2MNaHCO3, 0.5MNaCl, pH8.6

(coupling

buffer).

Themixture was rotated at room temperature for 2h and treated with 1 M etha-nolamine for 2h at roomtemperature, washed three times with sodium

acetate buffer (0.1 M, pH 4.0,

containing

0.5 sodium

chloride),

and alternated withaborate buffer(0.1 M, pH8.0,also

containing

0.5M

NaCl).Thematerialwasthen washed with thecouplingbuffer followed

100

80

C

0-GI

cu

qs

Serum dilution

by0.1 M glycine buffer, pH 8.0, followed by the coupling buffer, followed by PBS; then, it was placed in a 5-ml syringe, plugged with glass wool, and equilibrated with PBS at room temperature.

Absorption with

lysine-Sepharose

100 ml of normal human serum heated at 60°C for 1 h was applied to alysine-Sepharose 4B column (20 X 2 cm) previously equilibrated with 0.05 M phosphate buffer, pH 7.5, at4°C by using a flow rate of 20 ml/ h. The serum was assayed for plasminogen on LM-Partigen immuno-diffusion plates.

Fibrinogen and

fibrin

digestion

products

3 mgof fibrinogen contained in 1 ml of distilled water was incubated with 0.037 ml of plasmin (80IU/ml) at 18°C and the reaction stopped at 0, 1/2, 1, 4, and 24 h with 10 ul of aprotinin (20,000 KI units/ml). The samples were dialyzed against PBS and 50

_,ul

fractions were tested for GPBP activity. For fibrin, the same quantities of fibrinogen were used butthrombin (250,ulcontaining 5.0 IU/ml with 40 mmolCaCl2)

was addedfor 2 min before mixing with plasminogen.

Results

Fresh serum and serum heated at 56°C for 1 h, from nine normal healthyindividuals, were tested for their ability to pro-mote adherence of granulocytes to pollen grains (Fig. 1). A concentration-dependent increase of adherenceofneutrophils

to TGPwasobserved. There were no appreciable differences between the heated and unheatedsera. In other experiments, it was found that, in general, there were no

differences

between

anautologous system, i.e., serum and granulocytes from the same

donor,

or

experiments

in which cells and serum from different individualswere used.

When normal serum wasapplied to a column of Sephadex G-200, the major peak of granulocyte/pollen adherence eluted

together with proteins havingamolecular mass of

-70,000

D

(Fig.

2 A). Asmaller

peak

of

activity

wasalso observedatthe void volume

(VO),

although the degree

of

adherenceaswellas

the percentage adherencewas

considerably

less than that

ob-Figure1.Adherence of

granulocytes

to TGPbyheatedand unheated normal

serafromhealthyindividuals.NHS,

normalhuman serum;NHSH =serum

heatedat56°Cfor 1 h.

Granulocytes

andsera wereobtained from thesame

A

900K 150K 67K 1-35K

I- 5-- Pool

*1~~~~10

'SOC

00 06

I , , , , o <

80 100 120 140 160 180 200

2000 K

B

I

20-

15-10

0 5

-

0-iK 1 35K

Pool

Sephadex G-75

20 30 40

Fraction number

100

-80

-60

-40

20

0

Figure2.Gel filtrationchromatography. (A) SephadexG-200: the

experimentwasperformed 12times, i.e.,NHS(6)and NHSH(6).

Theexampleshown is NHSH.(B) SephadexG-75: the experiment

wasperformed 12times(asin Fig. 2A).

NHS, normal humanserum;NHSH,serum heatedat56°C for I

h;OD, optical density.

served with the peak eluting in the albumin region. The

ex-perimentwasperformedon 12occasions (six with freshserum,

six with heated serum). Theresults were virtually identical in

allinstances. The70,000-D peakwasthen pooledasindicated,

concentrated, andappliedtoacolumn of Sephadex G-75 (Fig.

2B).Asinglepeakofgranulocyte/pollen adherencewasobserved

whichelutedwithasingle peak of protein. By SDS-PAGE,this

material wasshown tocontain albumin and other proteins in the 60,000-80,000 molwt region together withtraces ofIgG.

This experiment wasalso performed 12 times (six with fresh serumandsix with heated serum) andgavevirtually identical

resultsoneach occasion.

Material prepared by Sephadex G-200 and G-75

chroma-tography was applied to a column ofBlue Sepharose. GPBP

was associated with the protein peak that did not bind. The

proteinthat boundtoBlueSepharose (identifiedasalbuminby

immunoelectrophoresis againstamonospecificantialbumin) and which eluted with 0.05 M

Tris/HCI,

pH 7.0, plus

1.5 M KCI containedno

pollen-binding activity.

Protein-containing GPBP

activity,

which did not bind to Blue Sepharose, gave anumber of faint bandson

SDS-PAGE,

including IgG.

Accordingly, this materialwas

pooled, dialyzed,

lyophilized, and reconstituted, and applied to a column of

protein

A-Sepharose. Virtually all the protein present, which also

con-tained GPBP activity, failedtobindtoprotein A-Sepharose. A small peak of IgG waseluted with I M acetic acid. This did not contain GPBP activity.

The characteristics of GPBPon anion exchange chroma-tographywere studied by using DEAE-Sephacel (Fig. 3). Two broad peaks of GPBP activity were observed in the material which did not bind to DEAE-Sephacel with 0.02 M

phosphate

buffer and 0.06 M NaCl, pH 7.8. Almost all of the GPBP activity wasdetected inthesecond peak which eluted immediately after IgG. Only a weak degree of adherence was observed with the first peak. The second peak was pooled as indicated and applied

to acolumn of G-200 (Fig. 4). GPBP activity wasclearly sep-arable fromthe single major protein peak which consisted mostly ofIgG. When a salt gradient was applied, no further GPBP activity was observed in the other proteins eluting from the column.

Further attempts to purify the first peak of GPBP activity (Fig. 3) by

Sephadex

G-200 gave inconclusive results since several peaks of weak GPBP activity were observed at several bed vol-umes.

GPBP was then progressively purified by using a combination ofDEAE-Sephacel, Sephadex G-200, Blue Sepharose, and pro-teinA-Sepharose affinity chromatography in sequence. In these further studies, the normal human serum was firstapplied to acolumn oflysine-Sepharose. This initial procedure resulted in no loss

of

GPBPactivity, but it depleted the unfractionated

serum of plasminogen by >90% as assessed by single radial immunodiffusion using a

monospecific

antiplasminogen.

This progressive purification was performed on five occasions. In eachinstance, the final material contained a protein which gave

abroad band on

SDS-PAGE (Fig.

5). The molecular weights of thesepreparations were all between 67,000 and 82,000, and had an

isoelectric

point (pl) between 5.5 and 6.1 (Fig. 6).

These purified preparations, which had GPBP activity in the concentration range of 10-50

Aig/ml,

were tested for the presenceof a number of recognized serum proteins. These in-cluded the C3b inactivator,

f3-2-glycoprotein

1, Factor

B,

C4

binding protein,

fibronectin, and serum amyloid P component. None of these proteins were detected in purified GPBP. The

C4 binding protein, serum

amyloid

P component, and fibro-nectin were measured by Dr. M. B. Pepys (Royal Postgraduate Medical School, Hammersmith Hospital, London), by using electroimmunoassay and monospecific antisera.

In

further

separatestudies, fibrin or fibrinogen were digested with plasmin at time intervals and the fibrinogen/fibrin digestion products were tested for GBPB activity. The digestion was

0

E

c C

0

0

E C:

co

0

DEAE

-

Sephacel

Pool

1.( E

S

E-C

40 (0

2 0

1*0-0*0

-0- 0

Fraction number

stopped at 30

min,

1, 4, and 24 h by the addition of aprotinin. NoGPBPactivity was observed in any of the samples tested. Purified lactoferrin, GPBP, and transferrin were tested for

gran-ulocyte/pollen-binding

activity at 12.5, 30, 125, and 300

_,4g/ml

onfour occasions. No activity was observed with lactoferrin at any

of

the concentrations tested and no lactoferrin was present in the GPBP or transferrin preparations, as assessed by im-munoelectrophoresis. In contrast, GPBP and transferrin gave

adose-dependent

increase

in

granulocyte/pollen binding with

70%(++)-80% (++) adherence with the

highest

doses.

2000K 150 K

'I

67K

Pool

-20

4

-15

-10LA

.4-0 U

-0

100

- 80

ou

- 60 0

-40

-c Figure 3. GPBP activity in 20 E normal serum separated by

n

DEAE-Sephacel

anion 0 <

exchange

chromatography.

The

experiment

wasperformed five times(3X

NHSH;

2XNHS).

OD,optical density.

Crossed-over

immunoelectrophoresis

was used to identify GPBP. A sample of GPBP was mixed with normal human

serumand

after

electrophoresis in two dimensions, was shown

tosubstantially increase a protein peak in the

if-1

region,which wasidentified as transferrin (Fig. 7). Furthermore, a monospecific antiserum which was raised against GPBP gave a line of identity with transferrin, and antitransferrin gave a line of identity with GPBP andtransferrin (Fig. 8). The anti-GPBP was then absorbed

on to

CNBr-activated

Sepharose particles and an

immunoab-sorption experiment performed

asshown in Fig. 9 A. A purified

1-35K

| Sephadex G-200

-100

-80 0

41

-60 '

do

40 Li

a)

F20

'

-0

Fraction

number

Figure 4. Sephadex G-200

chromatographyof GPBPpurified

byDEAE-Sephacel. The material fromDEAE-Sephacel(Fig.4)was

pooled,dialyzed,and

lyophilized

asindicated,andresuspended in

PBS, pH 7.35, andappliedto a

SephadexG-200(85X 5cm)

column withaflowrateof 30

ml/

h. 10-ml fractionswerecollected and theexperimentwas

performed

at4VC.Alternatefractionswere

tested for GPBP

activity

as

indicated. Theexperimentwas

repeated four times. OD,

optical

density.

1

0-0

E

c

C> Go

C3 CD

08

-0 6

-0-4

-0o2

-

Molecular

markers

_-, <4 330K

-

-

.

-f '. 67K

M- 4* 60K

- '- 36K

18-5K

A

B

C

D

FigureS. SDS-PAGE of GPBP and transferrin.A, GPBPpurified by DEAE-Sephacel, Sephadex G-200andSephadex G-75,Blue

Sepharose, andproteinA-Sepharose (+ ME);B, asA(withoutME); C, commercial transferrin (+ ME); D, molecular markers (+ ME). ME,mercaptoethanol.

preparation of GPBP, which gaveasingle band of SDS-PAGE, wasapplied to the column. There was noprotein or biological activity in the material which didnotabsorbtothecolumn. A

single protein

elutedfrom the

Sepharose

with 0.1Macetic

acid,

andthiswasshowntocontaina

single protein peak

containing

asingle peak of

granulocyte/pollen-binding

activity.

Thesame

experiment

was

performed

with antitransferrin coupledto Se-pharose4Band thesameresultswere

obtained,

although with this procedureavery small

protein

peak with minimal

biological

activity

was observed in material whichdid notadsorbtothe column

(Fig.

9 B).

Thematerial which adsorbed to and eluted

from

anti-GPBP gavea

single

lineon

immunoelectrophoresis against

anti-normal

I

;I

I.I

C> Ll co %Oso0 nL u -4

pH

Figure6.Isoelectricfocusingof GPBP. The

asin(Fig. 5A) withtheexception of protei

experimentwasperformedthreetimes.

human seruminthebeta-I region and against anti-GPBPand antitransferrin. The material which didnotadsorbtoanti-GPBP didnotgivealineagainst anti-GPBP, whereas GPBP and

trans-ferrin gaveidentical lines with antitransferrin and anti-GPBP. Having establishedthat GPBP and transferrinwereidentical, normal human serumwas saturated with ferric chloride before chromatography on DEAE-Sephacel. Onthisoccasion, onlya

single broad peak ofbiological activitywas observed whicheluted after IgG, i.e., the weak peak of activity which was observed when the same experiment was performed without iron satu-ration was not detected (Fig. 3). As before (Fig. 3), no further proteins having GPBP activitywereobserved after application ofthe salt gradient. The same experimentswere performed in which the 0.06 M NaCI wasomitted from the starting buffer. Transferrin and GPBP eluted together after the application of

the salt gradient (0.02-0.035 M NaCl). In the two instances

where it wasperformed, the percent recoveries of transferrin, as estimated by gel diffusion, were 81.3 and 84.7%. Virtually the same resultwasobtained when commercial transferrin,

sat-uratedwith iron, wasseparated by DEAE-Sephacel.

When either normal human serum saturated with iron, or

commercial transferrin saturated with iron, from DEAE-Se-phacel wereapplied to Sephadex G-200,single peaks of activity were observed whicheluted with a major protein peak. In turn, these cochromatographed with thesingle peak of transferrinas

assayed by rocketimmunoelectrophoresis. Unlike the resultsin

Fig. 2 A in which normal human serum was chromatographed without prior saturationwith ferric chloride, the smaller weaker peak of

activity

previously observed atV0was notobserved.

Purified

iron-saturated

GPBP gave a dose-dependentincrease

in both the percent adherence and the degree of adherence. Concentrations as low as 1.25 ,ug/ml gave significantly more

binding than the diluent control, whereasat 300 ,ug/ml, there

was -85% binding and (+++) adherence.

Discussion

The assay used in the present study is simple, reliable, and reproducible. It is essentially a rosette technique but is consid-erably easier to quantify than erythrocytes/leukocyte rosettes becauseofthe ease of visualization ofthe pollen/leukocyte com-plex.With strongreactions, there was multilayer adherence of granulocytestopollengrains. Thiswasreminiscentof the large numbers of adherent erythrocytes observed in certain eryth-rocytes/leukocyte rosettes andis possibly due to alterations in

7 F| Tgthe1 netmembrane charge

of

the

innermost

granulocytesbinding

0-4

4 4 4 4 ~~~tothe

pollen

grain which,

in turn, renders these cells more

"sticky"

and leadsto

agglutination

with otherleukocytes. Theinitial observations with GPBP indicated that it was a

O;

heat-stableprotein, and therefore, unlikely to be IgE or to be generatedby complement

activation.

Itis yettobeshownwith preparationwasprepared certainty that serum fromallergicindividuals who have elevated inA-Sepharose. The concentrations of

TGP-specific

IgE

and

IgG

antibodies

4,

Figure7. Theeffect of GPBPon theprofileofcrossed immunoelectrophoresis of normal human serum.Thepoint ofthe arrowindicatesthe

transferrin peak. Normal humanserum isontheleft side, normalhumanserumplusGPBP (preparedasin Fig. 5A)isontheright side.

Infact, in preliminary experiments (Sass-Kuhn, S. P., R.Moqbel,

andA. B. Kay,

unpublished observations),

itwasshown that serafrom patients

with

seasonal

allergic rhinitis

and

high

TGP-specific IgE had no more

granulocyte/pollen-binding

activity than normal serum. The gel

filtration experiments

suggested

that thebiological

activity

wasassociated with albumin (Figs.

2 A and B). However, it was possible to separate GPBP from

albumin by Blue Sepharose

affinity chromatography.

The protein

A-Sepharose

studies

indicated

that IgGwas

unlikely

tobe

in-volved and therefore, this affinity step, when

considered

together with the fact that atopic and nonatopic sera gave similar results (Fig. 1), makes it

unlikely

that anti-TGP

antibodies play

a role

in this adherence reaction. It was

possible

to separate GPBP

from the majority of plasma proteins by

DEAE-Sephacel

and this served asa

useful

initial

purification

step for

further

studies

Tf GPBP Tf

anti-Tf anti-GPBP

Figure 8. Single radial immunodiffusion of GPBP and transferrin with antitransferrinandanti-GPB. 5_ti containing 10,gof

transferrin and5 ulof 25 ugofGPBPwereusedasindicated.The antiserawereusedatavolume of 75 Ml.Theantitransferrinwas

diluted I in2, and this anti-GPBPwasused undiluted.

(Fig. 3). Thus,

by

the combination of anion

_{exchange (Fig. 3),}

gel

filtration

_(Fig.

_4),

and

affinity chromatography, together

with

immunoelectrophoresis

and

SDS-PAGE,

GPBPwasshown to

be a

67,000-82,000

mol wt

_{(3-1-protein (Fig. 5)}

witha

_pI

of between 5.5 and 6.1

(Fig. 6)

anda_{component of normal}serum.

Purified

preparations

of GPBPwerefree of detectableamounts

of

albumin,

C3b

inactivator, (32-glycoprotein 1,

Factor

B,

C4

binding protein,

fibronectin

_fragments,

serum

_amyloid

P

com-ponent, and lactoferrin.

By

crossed-over

immunoelectrophoresis

with whole human

serum,GPBP accentuated

_markedly

the

height

ofthe transferrin

peak

but hadnoeffecton the

height

or

intensity

ofanyother

peak

of

plasma

protein,

including

hemopexin,

which hasasimilar molecular

weight

and

charge

to transferrin

(Fig.

7).

With the

useof

monospecific

antiserato both GPBP and

transferrin,

it

was

possible

to

show, by single

radial

immunodiffusion (Fig.

8)

and

immuno-affinity chromatography (Fig. 9),

not

only

that GPBP and transferrin were identical but that GPBP

activity

coeluted with this

iron-binding

protein.

With the

knowledge

that transferrin and GPBPwere

ap-parently

thesame

protein,

further

purification procedures

were

undertaken withprior iron-saturation. Thisgave

single

peaks

of GPBP

_{activity, i.e;, activity}

in the

high

molecular

weight

region

of

Sephadex

G-200

(Fig. 2)

wasno

longer observed,

and neither wasthe earlier

peak

on

DEAE-Sephacel (Fig. 3).

We

interpret

these

findings

as

being

aconsequenceof size and

charge

heterogeneity

of transferrin

resulting

frompooriron saturation since itwas

previously

shown that the transferrin molecule is unstable intermsof its behavioron

DEAE-Sephadex

and

Se-phadex

G-200 withoutpriorsaturation with iron (5). We also observed

that,

with iron-saturated

starting material,

fractions

AL|EAnti-GPBP1

3

004

II~,

I-oo

,rE++-z

vl

v-v-E c2

o0-02_0-4 ± 16I

~

-0~~~~~~~~~~~~~~~

0 2 4 6 8 10 12 14 16 18 20 22 24 26

B

Atimnsferrin

0.18

I0-12

4-CN 0 06- +

0-0.00-L

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Fraction number

Figure 9. Absorption of GPBP by anti-GPBP and antitransferrin. (A) Anti-GPBP. The GPBP applied to the column was prepared as in A (Fig. 5). 1mgof GPBP in 1 ml was applied to the column and l-ml fractions were collected by gravity. The protein was eluted with 0.1

Macetic acid as indicated in Fig. 10 and alternate fractions were

testedfor biological activity following dialysis against PBS at4°Cfor 18h. Theexperiment was performed four times. A representative example is shown. (B) Antitransferrin. The GPBP applied to the

columnwasasin A (Fig. 5). Theantitransferrin-Sepharose 4B columnwasprepared in anidentical fashion with the exception that thecouplingbuffer was0.1 MNaHCO3, pH 8.3, containing 0.5 M

NaCl.Thecolumn size of theantitransferrin was 22X 1 cm and the flowratewas60 ml/h.2-mlfractions were collected and 2 ml of 1 mg/ml was applied. Other conditions were as for anti-GPBP.

Transferrin (siderophilin) isa

f,-l-glycoprotein

with a

mo-lecular weight variously estimated to be between 68,000 and

90,000

(6-8). The pIof iron-saturated transferrin is -5.4 but

this is higher (5.8) in iron-free buffer(which were the conditions used in Fig. 7). It is formed mainly in the liver but possibly

also inthe

reticuloendothelial

system. Two atomsof iron in the

ferric

formattach to one molecule of transferrin but the protein

also binds other metals, although much less firmly than iron

(9, 10). Normal plasma contains 240-280 mg of transferrin/ 100 ml but as much as 50-60% of exchangable transferrin is present in extravascular fluid including tracheo-bronchial

se-cretions, saliva, tears, cerebrospinal fluid, and urine (11-13).

Thereasonforthe variation in molecular size is unclear. In the

present

study,

the different

preparations

of GPBP had

differing

molecular sizes

(Fig. 2).

Lactoferrin,

which is found

mainly

in breast milk and other

secretions,

shares many

properties

with transferrin intermsof

iron-binding properties,

molecular

size,

and

charge. However,

in the present

study,

we were able to

show that

purified

lactoferrin did nothave GPBP

activity

and that our

purified

GPBP

preparations

were not contaminated with lactoferrin.

Anumberof transferrin variants have been described which show characteristic

peptide

maps

suggestive

ofdifferences in

primary

structure

(5,

14).

Atleast 20 variants of transferrinare

known inman.Weareyettodetermine whether these variants differ in GPBP

activity.

It seems

unlikely

that GPBP

activity

is

directly

related to the

iron-binding capacity

of transferrin. For

instance, apotransferrin

was as effective asiron-saturated transferrin in promoting

granulocyte/pollen binding (Kay,

A.

B.,

S.P.

Sass-Kuhn,

R.

Moqbel,

and J.

MacKay,

unpublished

observations).

It has been

suggested

thatthe two

iron-binding

sites on the transferrin molecule differ in their

iron-binding

properties,

and the

possibility

that

they

also have distinctive roles in irontransportand metabolism

points

tothe

complexity

ofthe

uptake

and release of iron and other metals

by

this

protein

(15,

16).

The existence of specific receptors for transferrin on the surface of human

reticulocytes ( 17)

and other cells and tissues

including lymphocytes (18)

and the

placenta

hasbeen established

(19). However,

itseems

unlikely

thatthis conventional transferrin

receptoris involved in the transferrin-induced enhancement of

granulocyte/pollen

adhesion described in the present

study,

since the reactionwas notinhibitable

by

amonoclonal antitransferrin

receptor (OKT9) antibody (Kay, A. B., S. P. Sass-Kuhn, R.

Moqbel,

and J.

MacKay,

unpublished

observations).

Although the primary function of transferrininmammalian metabolism is thetransportof iron from sites of absorption and

storage tosites of

utilization,

such asbone marrow of normal animals and the

placenta

ofpregnant

animals,

it is also

con-sideredtohave antimicrobial properties resulting from the avid capacity of this proteintobind iron andcompetefor this essential nutrient required for the growth ofmost bacteria, fungi, and viruses.The presentfindings indicatethatenhancement

ofgran-ulocyte/pollen binding isafurther function of transferrin that is unrelatedtoirontransport or toits antimicrobial

properties.

The widespreadextracellulardistribution of transferrinmaybe relevant to the role of this protein in the removal of certain organic matter,including pollen grains.

Acknowledgments

Wearegrateful to Dr. Marcella Contreas of North London Blood Trans-fusion Center for supplying normal human serum.

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