identification and characterization of the cell-associated binding protein for urinary trypsin...

Ž .Biochimica et Biophysica Acta 1383 1998 253–268

Identification and characterization of the cell-associated binding proteinfor urinary trypsin inhibitor

Hiroshi Kobayashi a,), Yasuyuki Hirashima a, Guang Wei Sun a, Michio Fujie b,Kiyoshi Shibata b, Satoshi Tamotsu c, Katsutoshi Miura d, Dan Sugino e, You Tanaka f,

Satoshi Kondo f, Toshihiko Terao a

a Department of Obstetrics and Gynecology, Hamamatsu UniÕersity School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-31,Japan

b Equipment Center, Hamamatsu UniÕersity School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-31, Japanc Department of 1st Physiology, Hamamatsu UniÕersity School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-31, Japand Department of 2nd Pathology, Hamamatsu UniÕersity School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-31, Japan

e Nissin Central Research Institute, Nissin Food Products, Kusatsu, Shiga 525, Japanf Diagnostics DiÕision, Chugai Pharmaceutical, Chuo-ku, Tokyo, Japan

Received 29 September 1997; revised 2 December 1997; accepted 9 December 1997

Abstract

Ž .Urinary trypsin inhibitor UTI inhibits not only tumor cell invasion but also production of experimental and spontaneousmetastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for

w x Ž .UTI on their cell surface. Kobayashi et al., J. Biol. Chem. 269, 1994, 20 642–20 647 . UTI binding protein UTIBP waspurified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis

Ž .and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein LP . LP wasidentified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by thereactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH -terminal domain of UTI. UTI and2

UTIBP are distributed uniformly in the cytoplasm andror over the cell surface of tumor cells and fibroblasts. The level ofstaining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggestthat the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan–hyaluronic acid aggregates, whichinteracts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment closeto tumor and fibroblast cell surface. q 1998 Elsevier Science B.V.

Keywords: Urinary trypsin inhibitor; Link protein; HI-8; Hyaluronic acid

Ž .Abbreviations: AN, acetonitrile; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; HA, hyaluronic acid hyaluronan ;HABP, HA binding protein; HI-8, carboxyl terminus of UTI; HPLC, high performance liquid chromatography; LP, link protein; PBS,phosphate-buffered saline; PVDF, polyvinylidine difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;TBS, Tris-buffered saline; TFA, trifluoroacetic acid; UTI, urinary trypsin inhibitor; UTIBP, UTI binding protein

) Corresponding author. Fax: q81-53-435-1626.

0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0167-4838 97 00215-X

( )H. Kobayashi et al.rBiochimica et Biophysica Acta 1383 1998 253–268254

1. Introduction

Ž .Urinary trypsin inhibitor UTI is a Kunitz-typew xprotease inhibitor present in serum and in urine 1–7 .

UTI inhibits various serine protease such as trypsin,a-chymotrypsin, granulocyte elastase, plasmin,cathepsin G and hyaluronidase. UTI secretes intoplasma by human hepatocytes as the light chain of

Ž .inter-a-inhibitor Ia I , which consists of two heavyŽ . Ž . w xchains HC1 and HC2 and one light chain UTI 7 .

In recent years, a number of studies have emergedinvestigating the effects of UTI on the function of

winhibition of tumor cell invasion and metastasis 8–x15 . UTI inhibits not only tumor cell invasion in an in

w xvitro assay 8,10–13 but also production of experi-mental and spontaneous lung metastasis in an in vivo

w xmouse model 9,14 . The anti-invasive effect is de-w xpendent on the antiplasmin activity of UTI 8 . The

synthetic UTI peptide, which inhibits plasmin activ-ity, showed mouse 3LL cell invasion inhibitory activ-

w xity 11 . UTI and the effective peptide inhibited tumorw xcell invasion through Matrigel 11 . However, UTI

did not inhibit tumor cell proliferation or the bindingof the cells to Matrigel. Also, UTI did not inhibitchemotactic migration of tumor cells to fibronectinw x10,12,13 . It is likely that UTI acts as a protease

w xinhibitor 7,8 . In addition, UTI is thought to act oninhibition of a certain pathway in signal transductionto trigger processes involved in cell invasion and

w xmetastasis 10–14 . The importance of protease in-hibitor events involving tumor cell-associated pro-teases led us to investigate the nature of human tumorcell surface receptor or binding protein for UTIŽ . w xUTIBP 15 . UTIBP could act as a cell-associatedbinding protein directly cross-linking UTI.

In the previous study, we have partially character-ized the UTIBP present on the tumor cell membrane

w xof SMT-cc1 cells 15 . The binding of UTI to SMT-cc1 cells appeared to be highly specific since it wasinhibited by excess amounts of unlabeled UTI. Cellsurface complexes of molecular weight 40 kDa and110 kDa were isolated that appear to function asreceptors or binding proteins for UTI. Other tumorcells failed to express UTIBP, indicating that theymay lack the appropriate receptors or the total bind-ing sites may be completely occupied by the endoge-nous UTI. The role of peptide sequences from eachUTI domain and their interaction with tumor cells

were investigated. UTI derivatized with biotin orFITC was taken up by tumor cells in a dose-depen-dent manner. This cell association was partially inhib-ited with a monoclonal antibody 4G12, which specifi-

Ž .cally recognizes NH terminus domain I of UTI.2

The binding was inhibited by fluid phase UTI, butŽ .not HI-8, COOH terminus domain II of UTI, sug-

gesting that UTI binds to cells through a site in thew xUTI domain I 13,15 .

In this paper, we have identified and characterizedthe putative receptors or binding proteins for UTI.We have developed and optimized a protocol for theisolation of the 40 kDa UTIBP from tumor cells. Thedetermined residues of UTIBP showed homology to

Ž . w xhuman cartilage link protein LP 16 . A functionalbinding assay revealed the specific interaction be-tween the NH -terminal fragment of UTI and LP.2

UTI and UTIBP are distributed uniformly in thecytoplasm andror over the cell surface of tumor cellsor fibroblasts. These results suggest that one of thecell membrane-derived UTI-associated binding pro-tein is the LP of proteoglycan–hyaluronic acid aggre-gates.

2. Materials and methods

2.1. Cells and culture

Human choriocarcinoma SMT-cc1 cells weregrown in monolayer cultures in RPMI 1640 contain-

Ž . w xing 10% fetal calf serum FCS 17 . Cells wereharvested at semiconfluency by washing plates once

Ž .with cold phosphate-buffered saline PBS , pH 7.4.Cells were pooled and spun at 1000 rpm for 5 min at

Ž 8 .48C. Washed cells 1=10 cells were resuspendedŽ .in 5 ml in lysis buffer see below .

Human uterine cervical tissues were obtained fromhysterectomy specimens within 3 h after surgery. Thetissue dispersion protocol was essentially identical tothat used to disperse skin to obtain dermal fibroblasts.In brief, the tissue was placed in HBSS at 48C andwas chopped into 1- to 2-mm fragments and washed.For dispersion, the fragments were incubated at 378Cwith 250 Urml collagenase, 120 Urml hyaluronidaseŽ .type 1-S; Sigma, St. Louis, MO , and 1%penicillin–streptomycin for 2 h. The partially di-gested tissue was filtered through nylon mesh of150-mm pore size. Dispersed cells were washed three

( )H. Kobayashi et al.rBiochimica et Biophysica Acta 1383 1998 253–268 255

times with PBS containing 0.1% BSA at 238C, andthe cells were then harvested. Cells were cultured in

Ž .Dulbecco’s modified Eagle’s medium DMEM withŽ .10% fetal calf serum FCS and maintained at 378C

in a humidified atmosphere containing 5% CO .2

2.2. Purification of UTI and UTI fragments

Ž .Urinary trypsin inhibitor UTI was purified tow xhomogeneity from human urine 8–15 . Elution of

purified UTI on a Sephacryl S-200 sizing columnindicated that the molecule migrated as an uncom-

Žplexed protein by molecular weight 50–60 kDa data.not shown . Sodium dodecyl sulfate-polyacrylamide

Ž .gel electrophoresis SDS-PAGE under nonreducingconditions showed that UTI migrated as a poly-

w xdisperse molecular weight 40 kDa band 8–15 . Byelectroblotting directly from SDS-PAGE, we identi-fied the one component on the basis of the aminoacid sequences. NH -terminal sequencing of the 402

kDa UTI preparation yielded the sequenceAVLPQEEEG, which corresponds to that reported

w xfor HI-30 1–7 . In addition, UTI was homogenousby SDS-PAGE prior to and after deglycosylation

Ž .with testicular hyaluronidase Sigma . It had an ap-parent molecular weight of 40 kDa prior to deglyco-sylation; this was reduced to 28 kDa after

w xhyaluronidase treatment 18–21 . Slight differencesin their reported molecular weight may be due tolaboratory to laboratory variations or to the extent ofthe glycosylation of the molecules.

The 30 kDa NH -terminal fragment of UTI and2Ž .the 8 kDa COOH-terminal fragment of UTI HI-8

were prepared from purified UTI by trypsin proteoly-w xsis as described previously 12,13 . The 30 kDa frag-

ment was not purified on trypsin–Sepharose, al-though it was quantitatively recovered in the flowthrough fraction. The split products from a trypticdigest of the NH -terminal UTI fragment were sub-2

jected to analytical SDS-PAGE under nonreducingand reducing conditions. Molecular characterizationof the fragments obtained by tryptic treatment of UTIwas analyzed. The NH -terminal amino acid se-2

quences, determined after electroblotting on PVDFŽ .sheets Millipore, Bedford, MA , of the NH -termi-2

nal UTI fragments were 1AVLPQEEEG and43FYNGTSMA. The 30 kDa fragment was not puri-fied to homogeneity. On the other hand, HI-8 re-

mained associated with the affinity matrix and wasw xpurified to homogeneity 12,13 .

A purified preparation of UTI was biotinylatedw xaccording to the method of Guesdon 22 , using

Ž .N-hydroxysucciminidyl biotinamidocaproate Sigmausing the manufacturer’s suggested procedures.

2.3. Isolation of the UTI binding polypeptide

Ž .Purified human UTI 5 mg or bovine serum albu-Ž .min BSA; Iwai Chemical, Tokyo, Japan; 5 mg were

Žcoupled to CNBr-activated Sepharose 4B 1.5 g dry. Žweights5 ml bed volume Pharmacia, Uppsala,

.Sweden according to the manufacturer’s recommen-dations. The procedure yielded greater than 99%

Žcoupling of proteins to Sepharose. Tumor cells 1=8 . Ž10 cells were lysed in 5 ml of lysis buffer 20 mM

Tris–HCl, pH 7.4 containing 150 mM NaCl, 1%Ž .wrv Triton X-100, 1 m grml testicular

w x.hyaluronidase, and 2 mgrml trypsin Sigma andincubated at 238C for 30 min. The resulting extract

Ž .was centrifuged 5000=g, 30 min, 48C and thesupernatant was mixed with BSA- or UTI-Sepharosebeads previously equilibrated in 20 mM Tris–HCl,pH 7.4, 150 mM NaCl, 0.2% Triton X-100, 10 mMbenzamidine, 1 mgrml leupeptin, 1 mM phenyl-methyl sulfonylfluoride, 0.1 mgrml ovalbumin, and

Ž .0.1 unitrml aprotinin all reagents from Sigma usingend to end rocking for 16 h at 48C. The affinity gelwas then washed 10 times with 20 mM Tris–HCl, pH7.4 containing 150 mM NaCl and twice with H O.2

Polyacrylamide gel electrophoresis under nonre-ducing conditions was performed using stacking gels

Ž .of 4% wrv acrylamide and a resolving gel of 15%Ž .wrv . A total of 50 ml of each Sepharose gelsolution was then dissolved in 50 ml of samplebuffer, followed by heating at 1008C for 3 min, after

Ž .electrophoresis, gels were stained with 0.1% wrvCommassie Blue R-250, destained, and rinsed with

Ž .H O. The visualized UTIBP band 40 kDa was cut2

from the gels with a razer blade and soaked for 30min in a solution of 20 mM Tris–HCl, pH 7.4, 0.1%Ž .wrv SDS with occasional swirling. Twenty gelslides were obtained by repeated experiments. Thegel slides were subjected to preparative gel elec-trophoresis. The component was isolated in a purestate after electroelution from SDS-PAGE; its relativemolecular mass was estimated as 40 kDa. The 110


kDa UTI binding protein was not collected. Elutedmaterials were concentrated by ultrafiltrationŽ .Amicon, Tokyo, Japan for analysis on reverse phase

Ž .high performance liquid chromatography HPLC .The C18 columns were packed for high performance

Ž .and equilibrated with 5% acetonitrile AN r0.1%Ž .trifluoroacetic acid TFA before loading. The mate-

rial was pumped directly onto the column. The col-umn was eluted at 1.0 mlrmin with a gradient from5% AN to 50% AN over 60 min. Fractions elutingbetween 28% and 31% AN were pooled and desig-nated UTIBP. Eluent was monitored at 214 nm.

2.4. Staphylococcal V8 protease treatment of UTIBP

UTIBP digestion was performed by mixing variousŽ .amounts of Staphylococcal V8 protease Sigma in

PBS at 378C for 30 min. UTIBP gave rise to splitproducts observed in the nonreduced digests at a V8proteaserUTIBP ratio of 1:50. At an enzyme sub-strate ratio of 1:10, UTIBP was almost completelydigested. The split products were characterized bySDS-PAGE. After separation, V8 protease-derivedforms of UTIBP were transferred to PVDF sheets.The visualized bands were cut from the sheets. NH -2

terminal amino acid sequence of the split productswere determined.

In a parallel study, a purified bovine nasal carti-lage LP was digested with V8 protease in PBS, pH7.4 for 30 min at 378C at a molar ratio of 1:10enzyme: substrate in order to follow the release oflow molecular weight protease-derived forms of LP.Aliquots were removed, denatured by boiling in 0.1%SDS, and subjected to gel electrophoresis and West-

Ž .ern blot see below .

2.5. Purification of boÕine nasal cartilage link pro-tein

Link protein was prepared after trypsin digestionof bovine nasal cartilage proteoglycans. The bovinenasal cartilage LP was purified by affinity chro-matography on HA covalently coupled to Sepharosew x23,24 . A purified preparation of LP was kindly

Ž .supplied by Chugai Pharmaceutical, Tokyo, Japan .

2.6. NH -terminal sequence2

NH -terminal amino acid sequence of the split2

products were determined using an automated protein

Žsequencer 475A, Applied Biosystems, Perkin-Elmer,.Urayasu, Japan equipped with an on-line HPLC

system for the detection of the amino acid phenyl-thiohydantoin derivatives, which were separated on aC18 column.

2.7. Solid-phase binding assays

Binding of UTI to the purified UTIBP proteinsŽ .UTIBP and UTIBP was measured by a solid-40 100

Žphase immunoassay. In brief, biotinylated UTI 0–.1000 nM was added to 96-well microtiter plate

Ž .Falcon, Oxnard, CA wells coated with the purifiedŽ . Ž .40 kDa UTIBP 2 mgrml , UTIBP 2 mgrml , or100

Ž .bovine nasal cartilage LP 2 mgrml for 2 h at 238C.Specifically bound biotinylated UTI was detected

Ž .with avidin–peroxidase 0.4 mgrml, 1 h, 238C . TheŽreaction with tetramethylbenzidine Cosmo Bio,

.Tokyo was terminated after incubation for 10 min atŽ .238C, and the A absorbance at 450 nm of each450

Žwell was measured in an EIA reader Model 2550,.Bio-Rad . Data are expressed as the mean"SD of

A of assays and carried out in triplicate. Back-450Žground binding to control wells wells coated with

.2% BSA was subtracted from each sample. Nonspe-cific binding was determined in the presence of a100-fold excess amount of UTI.

In addition, binding of biotinylated ligandsŽ .UTIBP , UTIBP , LP, or UTI; each 100 nM to100 40

the purified HA was measured by a solid-phase im-munoassay. Microtiter plate wells were coated with

Ž .the purified HA 100 mgrml for 16 h at 48C andthen analyzed as described above. The HA used inthe experiment was derived from chick’s comb and isa pure polysaccharide. HA was kindly supplied byChugai Pharmaceutical.

2.8. Preparation of rabbit anti-UTIBP IgG

A rabbit was immunized with 20 mg of UTIBP asŽan emulsion with Freund’s complete adjuvant Difco

.Lab. Detroit, MI . Three and four weeks after thefirst immunization, booster injections were per-formed. Two weeks after the last immunization, therabbit was bled and the antiserum was obtained. IgGfraction was purified by protein A–Sepharose column

Ž .chromatography Pharmacia .


2.9. SDS-polyacrylamide gel electrophoresis andWestern blot

Samples were analyzed by SDS-PAGE on a 12%acrylamide gel under nonreducing conditions. Fol-lowing polyacrylamide gel electrophoresis, the pro-teins were visualized by staining with CommassieBlue, or transferred to polyvinylidine difluorideŽ .PVDF paper using a semidry electroblotting appara-

Ž .tus Marysol, Tokyo . A rabbit polyclonal antibody toŽ .UTIBP 1:100 dilution was used as the primary

antibody. Following incubation with anti-UTIBP anti-bodies, the paper was washed and incubated in thesecondary antibody, goat anti-rabbit IgG conjugated

Žto biotin 1:500 dilution; Dako, Copenhagen, Den-.mark . This paper was washed again and incubated in

Ž .avidin–peroxidase 1:500 dilution; Dako and thenŽexposed to TBS containing 4-chloro-1-naphthol 0.6

. Ž .mgrml and hydrogen peroxide 0.02% , washed inw xdistilled water, and air-dried 10–15 .

2.10. Immunoprecipitation of UTIBP

The preparation of cell extracts and immunoprecip-itation of cell lysates with polyclonal antibody against

w xUTIBP have been described in detail elsewhere 15 .In brief, SMT-cc1 cells and fibroblasts were resus-pended in PBS containing D-biotinoyl-´-amino-

Žcaproic acid-N-hydroxysuccinimide ester biotin-7-.NHS according to the manufacturer’s instructions

Ž .Boehringer Mannheim . A total of 50 ml of anti-UTIBP antibody coupled to CNBr-activated

Ž .Sepharose 4B were incubated with lysates 500 mlof SMT-cc1 cells and fibroblasts. The final pelletsŽ .immobilized immunocomplexes were resuspendedin electrophoresis sample buffer and analyzed bySDS-PAGE, followed by Western blot using avidin–peroxidase.

2.11. Cell enzyme-linked immunosorbent assay for( )biotinylated UTI cell ELISA

Cell ELISA has been described in detail elsewherew x15 . In brief, human uterine fibroblasts were seeded

Ž .in 96-well microtiter plates Costar, Cambridge, MAand allowed to grow to confluence. To determine thespecific binding of biotinylated UTI to the surface ofthe cells, cells cultured in microtiter plates were

Ž .treated with biotinylated UTI 100 nM in the pres-

Žence of increasing concentrations of competitors un-labeled UTI, LP, mAb 4G12, pAb D2, anti-UTIBP,or nonimmune IgG; mAb 4G12 reacts with the NH -2

terminal domain I of UTI, while pAb D reacts with2wthe COOH-terminal domain II of UTI see Refs.

w xx. Ž13,15 for 1 h at 238C. Avidin–peroxidase 0.4.mgrml; 1 h, 238C was added to the wells, and the

peroxidase activity was determined in an EIA reader.All assays were carried out in triplicate.

2.12. Immunohistochemistry

Human uterine cervical carcinoma tissues wereobtained from panhysterectomy specimens. Permis-sion to conduct this study was obtained from theHamamatsu University Hospital Committee on Clini-cal Investigations. Samples of tumor tissues wereobtained from five patients treated at the Departmentof Obstetrics and Gynecology, Hamamatsu Univer-sity Hospital. The histologic diagnoses were squa-mous cell carcinoma, highly to moderately differenti-ated, stage IIb. No patient had received previoussystemic therapy or preoperative local radiotherapy.

The tissue pieces were fixed in solutions contain-ing 2% formaldehyde and 0.5% glutaraldehyde in 0.1M PBS. The paraffin blocks were cut to 5-mm sec-tions. The sections were dewaxed and rehydrated in agraded series of alcohol solution. In addition, thelocalization of UTI, UTIBP and hyaluronic acid wasstudied in the confluent nonpermeabilized SMT-cc1cells and human uterine cervical fibroblast cultures inchamber slides. The slides were treated with 10%H O for 5 min to eliminate endogenous peroxidase2 2

activity and then incubated for 16 h at 48C with 10mgrml of a rabbit anti-UTIBP antibody diluted inPBS containing 2% BSA. This was followed by a60-min incubation with FITC-conjugated anti-rabbit

Ž .IgG Dako diluted 1 to 100 in PBS, 2% BSA, andthen rinsed in two changes of PBS for 5 min each.The background staining for the UTIBP was deter-mined by substituting rabbit IgG for the rabbit anti-UTIBP IgG. This background staining was consis-tently negative. For histological comparison, somesections were stained with hematoxylin and eosin.

In a parallel experiment, the specimens were incu-bated for 16 h at 48C with a rabbit polyclonal anti-

Ž .body against human UTI 1 mgrml and a rabbitpolyclonal antibody raised against purified UTIBP40


Ž .10 mgrml , respectively. The sections were thenincubated for 30 min with FITC-conjugated anti-rab-bit IgG, diluted 1:125 with PBS containing 2% BSA.

Tissue samples from uterine cervical carcinomaŽwere frozen in liquid nitrogen. Cryostat sections 8

.mm were mounted onto slides and air dried. Some ofthe specimens were submitted to hyaluronidase diges-tion. Digestion conditions were 0.5 U of enzymermlin TBS, pH 8.0, containing 10 mM phenylmethylsulfonylfluoride at 378C for 1 h. Following digestion,the sections were washed in PBS and preincubated

Ž .with 2% BSA 20 min, 238C , briefly washed in PBS,Ž .and incubated with each antibody 1 h, 238C . After

staining with the primary antibodies, sections werewashed three times with PBS, incubated with FITC-

Žlabeled affinity-purified goat anti-rabbit IgG 60 min,.238C , washed three times with PBS, and examined

by fluorescence microscopy.Colocalization of HA was examined using the

Ž .FITC–conjugated HA binding protein FITC–HABPprobe for HA. The isolation of HABP has been

w xdescribed in detail elsewhere 25 . In brief, a mixtureŽof proteins having affinity for HA the HA-binding

region of the chondroitin sulfate proteoglycan and the.link proteins was isolated from bovine nasal carti-

lage and purified by affinity chromatography. Thepurified HA-binding region was then linked to FITCand stored at y208C until further use. Sections were

Ž .reacted with the FITC–HABP 5 mgrml dilutedwith 2% BSA in PBS for 16 h at 48C. After washingin PBS three times for 5 min each time, the sectionswere examined by fluorescence microscopy. As acontrol, some of the sections were reacted with nor-mal serum in place of the specific antibodies orprobe, and some were incubated with the FITC–HABP in the presence of an excess amount of HA.

3. Results

Direct binding experiments with biotinylated UTIin human choriocarcinoma SMT-cc1 cells have al-lowed us to identify a novel UTI receptor or UTI

w xbinding proteins on the cell surface 15 . It has beenreported that binding of UTI to the SMT-cc1 cellswas specific for the NH -terminal fragment of UTI2w x15 . UTI competed for the binding of the biotinyl-ated UTI to the SMT-cc1 cells in a dose-dependent

manner with half-maximal inhibition observed at ap-proximately 0.1 mM. In contrast, the COOH-terminalplasmin-inhibiting domain, HI-8, did not inhibit bind-

w xing 15 .

3.1. Purification of UTIBP

SMT-cc1 cells were solubilized in PBS containing1% Triton X-100, 1 mgrml hyaluronidase, and 2mgrml trypsin, and the soluble fraction was incu-bated with UTI-Sepharose. Bound material was ana-

Ž .lyzed by SDS-PAGE Fig. 1A . One major polypep-tide band was observed as a protein with molecular

Ž .weight of approximately 40 kDa Fig. 1A, lane 2 .This protein was subjected to preparative gel elec-trophoresis and was further purified by gradient elu-

Ž .tion on reverse phase HPLC Fig. 1B . The purifiedUTIBP revealed a single band of 40 kDa undernonreducing and reducing conditions on SDS-PAGE.We cannot calculate the yield and recovery, becausethe purification is not quantitative.

3.2. NH -terminal sequence of UTI binding protein2

ŽA sample of 25 pmol of the purified UTIBP Fig..1a, lane 3 was sequenced in an Applied Biosystems

475A gas-phase automated sequenator, and the com-plete amino acid sequence is shown in Table 1. Thisprotein exhibited a single NH -terminal sequence and2

the first 17 amino acids of UTIBP were analyzed.The determined residues of UTIBP showed homol-ogy with a sequence obtained from proteoglycan core

Ž . w xprotein of human cartilage link protein LP 16 .Ž . Ž .Residues 10 A and 15 P differ from those of LP

Ž . Ž .S and N, respectively . Residue 41 N of LP is asite of N-linked glycosylation, while UTIBP may notbe N-glycosylated. These data demonstrate that theNH -terminus of UTIBP does not correspond to the2

NH -terminus of full-length human LP.2

For determination and confirmation of the com-plete sequence of UTIBP, this protein was digestedwith V8 protease, and the resulting peptides were

Ž .separated Fig. 2 . In the course of determining theprimary structure of UTIBP, two peptides producedby digestion of UTIBP with V8 protease have beenselectively purified by SDS-PAGE. These poly-peptides have been sequenced and were identicalwith sequences obtained from LP. These data support


Fig. 1. SDS-polyacrylamide gel electrophoresis of UTIBP at various stages of purification. Characterization of UTIBP by SDS-PAGE inŽ .nonreducing gels. A UTIBP was purified from the SMT-cc1 cells as described under Section 2. The samples were characterized with a

Ž . Ž .12% SDS-PAGE gel. The detergent extraction of SMT-cc1 cells was mixed with BSA lane 1 - or UTI-Sepharose lane 2 . EachŽ .Sepharose gel solution was then dissolved in sample buffer. Lane 3, purified UTIBP 40 kDa . UTIBP was isolated in a pure state after

electroelution from preparative electrophoresis. The eluted material was concentrated by ultrafiltration. There is nothing visible in lane 1.The 40 kDa band in lane 2 is very faint and is unlikely to be clearly visible on reproduction. Lane 4, low molecular weight prestained

Ž . Ž . Ž .protein standards Bio-Rad . B A portion of the purified sample 40 kDa was applied to C18 column equilibrated in 5% ANr0.1%Ž .TFA and eluted at 1.0 mlrmin with a linear gradient from 5% AN to 50% AN. Peaks 1 and 2 filled rectangle are identified based on

Ž .their NH -terminal amino acid sequence analysis see Table 1 . Eluent was monitored at 214 nm.2

that the UTIBP and LP are similar polypeptide chainsthat are related to each other. Neither band B nor Cyields the NH -terminal sequence of band A. If2

bands B and C are the two cleavage products of bandA, one of them should be the NH -terminal amino2

acid sequence. There are additional bands on the gelthat could correspond to the NH -terminal peptide,2

Žsince we only sequenced two major bands B and C.on Fig. 2 . Bands B and C are clearly the result of

partial digestion, with B consisting of the COOH-terminal 2r3 of LP and band C being the COOH-terminal 1r2. The fuzzy bands above band C areprobably the NH -terminal 1r3. This data seems to2

be good proof that UTIBP is LP.40

The purified UTIBP was again digested with V8protease at a molar ratio of enzyme to substrate of1:200 at 378C for 12 h, and the resulting peptideswere separated by reverse-phase HPLC. Three majorpeptides were isolated, and each peptide was alsosequenced. The sequence of one of three peptides

was identical to that of the NH -terminal region of2

UTIBP.Rabbit polyclonal antibodies were raised against

the purified UTIBP, and the specificity of the anti-bodies towards UTIBP was confirmed by Westernblot analysis. The polyclonal antibodies obtained fromrabbits immunized with purified UTIBP reacted withpurified bovine LP. Correct interpretation of the se-quence data and crossreactivity of anti-UTIBP anti-bodies indicates that UTIBP is LP and not a homo-logue thereof. Western blot analysis of cell lysatesusing anti-UTIBP antibodies showed that these anti-bodies react with polydisperse band of around 100

Ž .kDa UTIBP and with a sharp band of approxi-100Ž .mately 40 kDa UTIBP as well as with minor40

Ž .components Fig. 3 .Fig. 4 shows that UTIBP and UTIBP proteins100 40

were eluted from cell lysates by affinity chromatogra-phy with the anti-UTIBP IgG coupled-Sepharose 4Bcolumn. Appropriate amounts of 100 kDa protein


Table 1NH -terminal sequence of UTIBP2

Ž .Cycle no. PTH Yield pmol

Amino acid

Fig. 1a, lane 3, 40 kDa: 88% homologyŽ .1 V V 8.32Ž .2 E E 7.69Ž .3 A A 5.03Ž .4 E E 7.08Ž .5 Q Q 6.31Ž .6 A A 5.00Ž .7 K K 6.14Ž .8 V V 5.35Ž .9 F F 4.17Ž .10 A S 4.32Ž .11 H H 2.14Ž .12 R R 1.97Ž .13 G G 4.21Ž .14 G G 3.98Ž .15 P N 3.87Ž .16 V V 3.68Ž .17 T T 1.09

Fig. 1b, peaks 1 and 2: 88% homologyŽ .1 V V 9.58Ž .2 E E 8.95Ž .3 A A 6.23Ž .4 E E 8.34Ž .5 Q Q 7.60Ž .6 A A 6.26Ž .7 K K 7.81Ž .8 V V 6.12Ž .9 F F 5.07Ž .10 A S 5.84Ž .11 H H 3.29Ž .12 R R 2.99Ž .13 G G 5.12Ž .14 G G 5.08Ž .15 P N 4.91Ž .16 V V 4.70Ž .17 T T 2.15

Fig. 2, lane 2-B: 100% homologyŽ .1 G G 9.24Ž .2 L L 6.43Ž .3 E E 7.06Ž .4 D D 6.61Ž .5 D D 6.50Ž .6 T T 2.05Ž .7 X V 4.65Ž .8 V V 4.58Ž .9 V V 4.50Ž .10 A A 4.78

Ž .Table 1 continued

Ž .Cycle no. PTH Yield pmol

Amino acid

Fig. 2, lane 2-C: 100% homologyŽ .1 A A 6.95Ž .2 Q Q 5.35Ž .3 Q Q 5.69Ž .4 A A 5.58Ž .5 X C N.Q.Ž .6 L L 3.48Ž .7 D D 5.00Ž .8 Q Q 3.67Ž .9 D D 3.76Ž .10 A A 3.31

Ž .1 Q Q 6.17Ž .2 Q Q 5.35Ž .3 A A 4.20Ž .4 X C N.Q.Ž .5 L L 3.03Ž .6 D D 4.18Ž .7 Q Q 3.78Ž .8 D D 3.43Ž .9 A A 3.12Ž .10 V V 3.32

NH -terminal amino acid sequences of UTIBP and V8 digestion2

products. UTIBP and two peptides derived from V8 proteasedigestion were subjected to NH -terminal amino acid sequence2

analysis. Undetermined amino acid residues are represented as X.Percents of identical amino acid residues in each comparison are

Ž .shown. , the corresponding amino acid sequences of the humanLP for comparison. The VEAE sequence represents position 27of native LP, GLED represents position 129 and EAQQ repre-sents position 161. The stepwise yields of all the PTH-aminoacids were included in the table. Approximate yields of phenyl-thiohydantoin derivatives were calculated from peak areas onHPLC. The values are background corrected. The purified UTIBP

Ž . Žused was 25 pmol Fig. 1a and b . Each peptide B and C.approximately 20 pmol was also sequenced in a gas-phase

sequenator. N.Q., not quantitated.

produced during one step purification were still ob-served even in this preparation.

Ž . ŽUTIBP Fig. 5A, lane 2 and UTIBP Fig. 5B,100 40.lane 2 were separately purified from tumor cell

lysates through two consecutive steps including anti-UTIBP antibody-coupled affinity chromatography andSephacryl S-200 gel filtration. Sugar chains of theUTIBP were analyzed by digestion with100


Fig. 2. Staphylococcal V8 protease digestion of UTIBP. UTIBPŽ . Ž .5 mg; lane 1, A was digested with V8 protease 0.1 mg for 30min at 378C in 100 mM Tris–HCl, pH 8.0. The digested samplesŽ .lane 2, B and C were applied to SDS-PAGE and transferred to

Ž .PVDF sheets. The sheets were stained with 0.1% wrv Com-massie Blue R-250, destained, and rinsed with H O. The visual-2

ized peptide bands were cut from the sheets. The NH -terminal2

amino acid sequence of each peptide was analyzed with a ProteinŽ . Ž .Sequencer Applied Biosystems see Table 1 .

Fig. 3. SDS-PAGE and Western blot of tumor cell lysates withŽ 7 .the anti-UTIBP antibody SMT-cc1 cell lysates 1=10 cells

Ž .were analyzed by SDS-PAGE 12% gel under nonreducingŽ . Ž .conditions Commassie Blue staining lane 3 . After SDS-PAGE,

proteins in the gel were transferred onto a PVDF membrane andWestern blot analysis were performed with the anti-UTIBP IgG.

Ž .Immunoblotting with rabbit anti-UTIBP IgG was done lane 4 .ŽLane 5, LP isolated from bovine nasal cartilage 1 mg; Western

.blot using anti-UTIBP antibody . Lanes 1 and 6, prestained highŽ .molecular marker proteins 213, 119, 83 and 47 kDa . Lanes 2

Žand 7, prestained low molecular marker proteins 108, 86, 51, 34,.and 27 kDa .

Fig. 4. Purification of UTIBP from tumor cell lysates by affinitychromatography with the anti-UTIBP antibody-coupled Sepharose

Ž 8 .4B column. SMT-cc1 cells 1=10 cells were lysed in lysisbuffer as described in Section 2. The resulting extract wascentrifuged and the supernatant was subjected to affinity chro-matography. The column was washed with 0.1% Triton X-100

Ž . Ž .and PBS totally 300 ml , followed by 1 M NaCl 50 ml , thenbound proteins were eluted with 0.2 M glycine–HCl, pH 3.0.Purified protein was dialyzed with distilled water, and lyophyl-

Ž .ized. Purified protein was analyzed by SDS-PAGE 12% gelunder nonreducing conditions and stained with Commassie Bril-

Ž .liant Blue R-250 lane 2 . Lane 1, prestained low molecularŽ .marker proteins 108, 86, 51, 34, and 27 kDa .

hyaluronidase followed by SDS-PAGE and WesternŽ .blot analysis with the anti-UTIBP IgG Fig. 5 .

Hyaluronidase digestion of UTIBP protein resulted100

in the appearance of degradation products of smallerŽ .sizes with the diffuse appearance Fig. 5A, lane 4 .

Extensive digestion yielded a component of around40 kDa. A doublet at about this molecular weight isclearly visible. Trypsin digestion of UTIBP also100

resulted in the appearance of degradation productsŽ .with the diffuse appearance Fig. 5A, lane 3 . How-

ever, more extensive digestion of UTIBP with40

hyaluronidase did not result in the appearance ofdegradation products of smaller sizes.

We reported that the NH -terminal amino acid2

sequence of the UTIBP was identical to that of the100Ž .UTIBP VEAEQAKVFAHRGG . The UTIBP is40 100

probably a complex form of the UTIBP and uniden-40

tified several sugar chains including HA androrchondroitin sulfate, since hyaluronidase will also di-

Žgest other glycosaminoglycans e.g., chondroitin sul-.fate in addition to HA. This may be the reason why


Fig. 5. Enzymatic digestion of the 100 kDa and the 40 kDaŽ . Ž .UTIBP proteins. A The purified 100 kDa protein 5 mg was

incubated for 30 min at 378C with the indicated amount ofŽ . Ž .trypsin lane 3, 0.1 mg , or hyaluronidase lane 4, 0.2 mg ,

respectively. Lane 1, low molecular weight prestained markerŽ .proteins Bio-Rad ; lane 2, the purified 100 kDa protein

Ž .UTIBP , 5 mg . Samples were analyzed by SDS-PAGE fol-100Ž .lowed by Western blot with the anti-UTIBP antibody. B The

Ž .purified 40 kDa protein 2 mg was incubated for 2 h at 378CŽ .with hyaluronidase 0.2 mg . Lane 1, low molecular weight

Ž .prestained marker proteins Bio-Rad ; lane 2, the purified 40 kDaŽ . Ž .protein UTIBP , 2 mg ; and lane 3, the UTIBP 2 mg treated40 40

Ž .with hyaluronidase 0.2 mg . Samples were analyzed by SDS-PAGE followed by Western blot with the anti-UTIBP antibody.

treatment with hyaluronidase generates the 40 kDaUTIBP. It seems more likely that UTIBP is a100

differentially glycosylated and highly heterogenousform of UTIBP .40

We further studied the direct binding of theUTIBP and the UTIBP to UTI. LP was purified100 40

to homogeneity from bovine nasal cartilage and wasused for a comparative study. UTI and its domain IIfragment HI-8 were biotinylated and used as ligands.A solid-phase binding assay was developed in whichUTIBP , UTIBP and LP were coated onto mi-100 40

crotiter wells and incubated with the biotinylatedŽ .ligands Fig. 6 . Specific binding of biotinylated UTI

was observed on UTIBP -, UTIBP -, or LP-coated100 40

wells, but biotinylated HI-8 did not bind to thesewells. To confirm the binding specificity of theUTIBP, a series of solid-phase binding experimentswere carried out with increasing concentrations of

Ž .soluble competitor proteins Fig. 7 . The binding ofŽ .the biotinylated UTI 200 nM to UTIBP - or100

UTIBP -coated wells decreased in a dose-dependent40Ž .manner in the presence of soluble UTI 0–1000 nM ,

Ž .but not in the presence of HI-8 0–1000 nM . TheID was approximately 100 nM for UTI. Using the50

Ž .same assay system, soluble LP 0–1000 nM wasalso tested for its ability to inhibit the binding of thebiotinylated UTI to UTIBP - or UTIBP -coated100 40

Ž .wells Fig. 7 . Soluble LP inhibited binding in a

Ž .Fig. 6. Direct binding of the biotinylated ligands UTI and HI-8 to immobilized solid-phase UTIBP proteins. Binding of the increasingŽ w x w xconcentrations of biotinylated UTI or biotinylated HI-8 to the adsorbed proteins UTIBP A , UTIBP B , or bovine nasal cartilage LP100 40

w x.C was determined at 238C as described in Section 2. LP was purified to homogeneity from bovine nasal cartilage proteoglycans for acomparative study. Results are the mean values"SD for triplicate determinations.


Fig. 7. Specificity of inhibition of the biotinylated UTI binding to immobilized UTIBP proteins by competitors. Binding of theŽ . Ž w x. Ž w x.biotinylated UTI 200 nM to microtiter wells coated with 100 kDa UTIBP 1 mgrml A or with 40 kDa UTIBP 1 mgrml B was

Ž . Ž . Ž .determined in the presence of the indicated concentrations of UTI v , HI-8 ` , or bovine LP I in solution. Results are meanvalues"SD for triplicate determinations.

dose-dependent manner. The ID for LP was 20–5050

nM. Based on the data from these solid-phase bindingassays, the UTIBP and the UTIBP polypeptide100 40

proteins appeared to function as a UTIBP specific forthe NH -terminal domain of UTI.2

Since it has been reported that LP bound specifi-Ž .cally to hyaluronic acid HA under physiological

condition, we examined whether UTIBP and100Ž .UTIBP have the same biological function Fig. 8 .40

A solid-phase binding assay revealed that the bio-tinylated UTIBP and UTIBP as well as biotinyl-100 40

ated LP bound specifically to immobilized HA in a

Fig. 8. Specific binding of the biotinylated ligands to immobi-lized hyaluronic acid. Binding of the increasing concentrations of

Ž . Ž .biotinylated UTIBP ^ , biotinylated UTIBP I , biotinyl-100 40Ž . Ž .ated bovine nasal cartilage LP ' , or biotinylated UTI v to

immobilized hyaluronic acid was determined at 238C for 2 h.Results are the mean values"SD for triplicate determinations.

dose-dependent and saturable manner, while UTI didnot bind directly to immobilized HA.

3.3. Human uterine fibroblasts and choriocarcinomaSMT-cc1 cells possess UTI-binding sites

The finding that UTIBP shows structural homol-ogy to the LP prompted us to investigate whether theUTI-binding activity expressed on fibroblasts wasrelated to LP. Subconfluent cultures of human uterinecervical fibroblasts and human choriocarcinomaSMT-cc1 cells were tested for their ability to bindbiotinylated UTI but only data for the fibroblasts areshown in Fig. 9. It was clear that the UTI-bindingactivity expressed on SMT-cc1 cells were less pro-

Ž .nounced than that in the fibroblasts data not shown .Two cell types exhibited cell surface UTI binding

Ž .sites. The binding of biotinylated UTI 100 nM wasinhibited by 90% or by 50% in the presence of 1000nM unlabeled UTI or in the presence of 1000 nM LPŽ .Fig. 9A .

Since MAb against UTI domain I, 4G12, inhibitedthe binding of biotinylated UTI to fibroblasts, 4G12was examined at different concentrations for their

Ž .ability to block UTI-binding activity Fig. 9B . Con-trol was a polyclonal antibody against UTI domain II,D2, which does not affect UTI binding. D2 reacts

Ž .with the COOH-terminal fragment of UTI HI-8 .Polyclonal antibody to UTIBP was also used to blockUTI-binding activity. 4G12 or polyclonal antibody to


Ž . Ž .Fig. 9. Effects of UTI and LP A and the specific antibodies B on the binding of biotinylated UTI to human uterine fibroblasts. Thebinding of biotinylated UTI to human uterine fibroblast cultures was performed in the presence of 100 nM of biotinylated UTI and

Ž . Žvarious amounts of competitors UTI or LP or antibodies nonimmune IgG, polyclonal antibody D2, polyclonal antibody to UTIBP, or.monoclonal antibody 4G12 . Each competitor or antibody was mixed with biotinylated UTI, then added to fibroblasts culture.

Biotinylated UTI binding was measured after 2-h incubation. Mean"SD of triplicate results are shown.

Ž .UTIBP 200 nM blocked about 70% or 40% of theUTI-binding activity, respectively, while D2 had onlymodest inhibitory effect on the binding activity. Al-though a major part of the UTI-binding sites onfibroblasts are related to LP, we cannot exclude thepossibility that other UTI-binding sites than the LPare present on fibroblasts.

3.4. Localization of UTIBP, UTI and HA

Cryostat sections were obtained from human uter-ine cervical carcinoma. By indirect immunofluo-rescence staining using anti-UTIBP antibodies, thelocalization of UTIBP was studied. To verify theobservation that UTI is expressed in the UTIBP-posi-tive cells, we examined the expression of UTI to seeif it colocalizes with cell-associated UTIBP. In addi-tion, since UTIBP bound specifically to HA, weexamined whether UTIBP colocalizes with the HA-positive cells. The localization of HA, UTIBP andUTI was compared in paraffin sections of humancervical carcinoma of the uterus.

All samples were stained with FITC–conjugatedHABP for HA as well as with polyclonal antibodies

Ž .against UTI and UTIBP Fig. 10 . In the cervicalcarcinoma of the uterus, HA was evident in thesurrounding tumor nests. Malignant cells containedno HA. HA was also evident in the perivascularzones of blood vessels. The perivascular HA was

particularly prominent in the adventitia, and occa-sionally extended to regions immediately beneath the

Ž .endothelium data not shown . Clearly the level ofŽstaining is much lower in Fig. 10B hyaluronidase

.treatment than in Fig. 10A. The HABP binds to HAand LP. The reduced level is due to the removal ofHA and LP associated with it. Therefore it is likelythat the weak staining after hyaluronidase treatmentis explained by the removal of HA and LP. There isconsiderably more staining in Fig. 10B than in Fig.

Ž .10C addition of exogenous HA . Therefore, there isstill weakly positive staining with biotinylated HABPeven after hyaluronidase treatment. A possible reasonfor this is that incomplete removal of HA has beeneffected.

In all specimens, UTI and UTIBP were also widelyŽobserved in the surrounding connective tissues Fig.

.10 . Tumor cells did not show definite reactivity.Tumor cells at the leading edge of invasively grow-ing tumor nests adjacent to the stromas showed veryweak UTI and UTIBP staining, and strong stainingwas present in the stromal cells at the leading edge oftumor nests. The distribution of UTI and UTIBP inthe uterine carcinoma tissues was quite reminiscentof that of HA, which was also located in the sur-rounding tumor nests. The distribution of HA, UTIand UTIBP was similar in different areas of thetumor infiltrates. Observation by serial sections re-vealed that the area of UTI and UTIBP reactivities in


Fig. 10. The distribution of HA, UTI and UTIBP in frozen sections of uterine cervical carcinoma. The specimens of cryostat sections areŽ . Ž . Ž . Ž . Žstained for HA A , UTI D , and UTIBP E using FITC–conjugated HABP 5 mgrml, 48C, 16 h , polyclonal antibody against UTI 1

. Ž .mgrml, 48C, 16 h , and polyclonal antibody raised against purified UTIBP protein 10 mgrml, 48C, 16 h , respectively. Staining for40Ž .HA is confined to stromal elements A . HA was observed surrounding tumor nests. HA labeling of the neoplastic cells is not observed.

Ž .The staining with FITC–conjugated HABP was completely eliminated by incubating the sections with hyaluronidase B or by theŽ . Ž .addition of exogenous HA C . The staining pattern seen in sections stained with biotinylated HABP A is similar to the staining pattern

Ž . Ž .seen in D and E. Staining for UTI D and UTIBP E is also confined to stromal elements. The control section stained with an antibodyŽ .of irrelevant specificity is negative data not shown . ‘Ca’ means cancer tissue.


Fig. 11. Immunoprecipitation of cell lysates with polyclonalantibody against UTIBP. Analysis of the molecular size of cell-associated UTIBP in choriocarcinoma SMT-cc1 cells and in theprimary cultures of fibroblasts. Immunoprecipitated samples wereanalyzed on a 12% SDS-polyacrylamide gel under nonreducingconditions and electroblotted prior to total protein staining. Celllysates treated with biotin-7-NHS were precipitated with poly-clonal antibody reacting with the UTIBP. Visualization wasperformed by using the avidin–peroxidase detection system.

Ž .Numbers at left lane 1 indicate molecular weight in kiloDaltonsŽ .kDa . Presented here is a representative Western blot from fourindependently performed experiments in which comparable re-sults were obtained. Densitometric scanning was performed toobtain the results of the 40 kDa UTIBP band from the representa-

Ž . Žtive Western blot of SMT-cc1 cells lane 2 and fibroblasts lane.3 . The 100 kDa band and the 40 kDa band in lane 2 are unlikely

to be visible on reproduction.

uterine cervical carcinoma was the same as that ofdiffuse deposition of HA.

Control staining experiments in which the specificantibodies were omitted or substituted with nonim-mune IgG were negative. Absorption of the FITC–conjugated HABP with highly purified preparationsof HA also completely abolished the staining of thetissues.

We further studied whether UTI and UTIBP arecolocalized on the same cell surface in the confluentnonpermeabilized SMT-cc1 cells and human uterine

Ž .fibroblast cultures data not shown . Immunocyto-chemical staining for HA, UTIBP and UTI was pre-sent uniformly over the cell surface of these cells. Wealso investigated whether the UTIBP located on thecell surface is sensitive to enzymes that degrade HA.

Hyaluronidase treatment appeared to strip most ofUTI and UTIBP from the cell surface as judged byhighly reduced immunostaining for UTI and UTIBP

Ž .after this treatment data not shown . Digestion byhyaluronidase may result in the release of UTI bind-ing elements. These results favor LP as the predomi-nant UTI ligand on the cells, since UTI does not bind

Ž .to HA directly Fig. 8 .In order to quantify the UTIBP on the cells we

analyzed biotin-labeled cell lysates by immuno-blotting using polyclonal antibody to UTIBP as a

Ž .probe Fig. 11 . Differential expression on fibroblastsand SMT-cc1 cells of UTIBP with the broad band ofmolecular weight )100 kDa and the sharp band ofmolecular weight 40 kDa were revealed. Densitomet-ric scanning showed that UTIBP was examined in40

low amounts on SMT-cc1 cells, corresponding toabout 20% of the UTIBP level found on the fibrob-lasts.

4. Discussion

We have previously identified tumor cell mem-brane proteins of molecular weight 40 kDa and 110kDa that bind UTI in the NH -terminal domain-de-2

w xpendent and specific manner 13,15 .In this paper, we have described the isolation and

characterization of the 40 kDa and approximately 100Ž .kDa UTI-binding proteins UTIBP and UTIBP40 100

from human choriocarcinoma SMT-cc1 cells anduterine fibroblasts using a UTI-Sepharose columnand preparative SDS-PAGE. Results of partial pep-tide sequencing indicate that this protein is essentiallyidentical to the truncated form of human cartilage

Ž . Ž .link protein LP Figs. 1 and 2, Table 1 . LP wasidentified structurally by its apparent molecular mass

Žwith and without deglycosylation treatment Figs. 1,.2, 4 and 5 : Immunologically by the reactivity with

Ž .anti-UTIBP antibody Fig. 3 , and functionally by itsability to bind the NH -terminal domain of UTI2Ž .Figs. 6–9 . The UTIBP should be composed of40

about 350 amino acids as estimated from the molecu-lar mass of this protein. The UTIBP probably shows40

considerable homology with the whole molecule ofLP composed of 354 amino acids. The 40 kDa pro-tein is claimed to be a link protein based on sequencehomology of peptides purified with a human link


protein from cartilage. However, it is not clearwhether this binding protein is identical to the humancartilage LP since only a partial sequence analysishas been obtained. The major difference is that a newprocessing site is used, resulting in an NH -terminal2

that differs from the forms described by other investi-w xgators 16,26 . In fact from the data provided in Table

1, it appears that the UTIBP first NH -terminal amino2

acid corresponds to amino acid 27 of link protein.LP is rather heterogenous, since it exists in a

number of forms, differing in molecular weight andcharge. These differing forms also result from differ-ing levels of glycosylation and from loss of a portionof the NH -terminal. Cartilage from different species2

or different age groups within the same species con-tains different amounts of three closely related glyco-

w xproteins, LP1, LP2 and LP3 16 . UTIBP is mostsimilar to LP3, which is a truncated form of LP thatis found in human cartilage in increasing amountswith age or can be produced in vitro by treatmentwith trypsin. In UTIBP, the glycosylation site atresidue 6 missing as a result of protease treatment.However, to date, the NH -terminus of UTIBP does2

not correspond to the NH -terminus of full-length2

LP. It is possible that UTIBP and LP are highlyhomologous and the specific UTIBP identified inneoplastic cells may differ from nonneoplastic cells.Thus, UTI isoforms may be involved in the malignantprogression of human tumors. Purification and char-acterization of fibroblast-derived UTIBP is now inprogress in our laboratory.

In addition, the UTIBP may have an additional100Ž .sugar chain besides the LP-like region Fig. 5 . A

possible candidate corresponding to this additionalregion is the HA andror chondroitin sulfate, suggest-ing that the UTIBP may be a LP-glycosamino-100

glycan complex. It is difficult to see, however, howUTIBP could be a LP-glycosaminoglycan com-100

plex. This would dissociate in SDS. However, it hasthe same molecular weight in SDS-PAGE both in thepresence or absence of mercaptoethanol. It is possiblethat this is a very highly modified form of LP.UTIBPs described here are antigenically distinct fromother well characterized cell matrix associated pro-

Žteins such as fibronectin, collagen, and laminin data.not shown .

Several types of cell surface molecules may beproposed as UTI receptors or UTI binding proteins in

w xvarious human cells 26 . It has been demonstratedthat UTI has a biphasic effect on fibroblast growth.Two types of cellular binding sites for UTI have beenidentified which may be responsible for the stimula-

w xtory and inhibitory aspects of this effect 26 . Wepropose that LP may constitute one of the majorbinding proteins for UTI.

A surprising finding is the localization of the UTIin stromal cells in the uterine cervical carcinomas,i.e., fibroblast cells, and the absence of UTI stainingin the cancer cells. A possible explanation for theincreased number of the UTI-positive cells in sur-rounding tumor nests is that they provide endogenousUTI needed for inhibition of tumor cell invasion. Thecancer cells may secrete substances that either attractthese UTI-containing cells from other parts of thebody or induce UTI synthesis in stromal cells. Itshould be noted that the UTI-positive cells do notnecessarily produce UTI itself. It is possible that UTI

Žis produced and released from one cell type hepato-.cytes and subsequently bound to the UTIBP on

Ž .another cell type stromal cells around tumor nests .UTI is produced and secreted into conditioned

media as an active form by human hepatocytes. Also,UTI can be produced in vivo by limited proteolysis

Ž .of inter-a-trypsin inhibitor ITI . ITI is comprised ofthree genetically different polypeptides, two heavy

Ž .chains and one light chin UTI . Although elucidationof the genuine roles of ITI family members is stilleagerly awaited, it has been reported that HA synthe-sized by cultured fibroblasts firmly bound the heavychains of ITI, which serves as a stabilizing factor for

w xthe extracellular matrix 27–31 . The heavy chains ofITI could interact with HA, while UTI did not di-rectly bind to HA. The domain structure of ITIsuggests that these molecules can interact with cellsand the extracellular environment in multiple ways.

We postulate the following process: UTI and ITIare produced in the liver and secreted into the serum,and then ITI is captured on the target cell surfacethrough the noncovalent or covalent linkage to HA

w xwith high affinity 32 , thereby mediating the bindingof HA to the cell surface and stabilizing pericellularmatrix. It is possible that a covalent interaction be-tween ITI and HA could result in this observed

w xincreased stability of the extracellular matrix 32 .Then, cell-associated ITI may be cleaved into UTI

w xand heavy-chains on the cell surface 33 . A part of


UTI is secreted into the serum and is excreted intothe urine. On the other hand, some of the UTImolecules are taken up on the pericellular matrix viaa HA-bound UTIBP or LP.

In the present study, expression of UTIBP in hu-man fibroblasts and human choriocarcinoma cells

Ž .was demonstrated Fig. 9 and expression of UTIBPin stromal cells surrounding tumor cells in specimenof human cervical carcinoma of the uterus was shownŽ .Figs. 10 and 11 . Binding studies on immobilizedHA indicate that the two UTIBP proteins bind to HA

Ž .at concentration of 100 nM Fig. 8 , although UTIcannot directly bind to HA. These data support ourhypothesis that cell membrane derived UTI bindingproteins are LP isoforms which interact with HA andmediate the binding of UTI to HA on the cell surface.These complexes may modulate the protease activityat the cell surface.

Our data not only suggest that each component ofITI may have a distinct functional role in cell behav-ior but also that distinct binding proteins or receptorsmay mediate these actions. By the identification andinitial characterization of UTIBPs, which may beidentical with LP isoforms, the analysis of the role ofUTI, which plays in cellular events such as proteaseinhibition and modulation of calcium influx or signal

w xtransduction, will be facilitated 34 .

Acknowledgements

We wish to thank Dr. E.E. Maradny for his adviceand critical review of the manuscript. We are gratefulto Drs. Tanaka and Kondo, Chugai Pharmaceutical,Tokyo, Japan for the generous supply of the HABP.

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identification and characterization of the cell-associated binding protein for urinary trypsin...

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