©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Fibronectin-binding Domain of Transglutaminase (*)

(Received for publication, November 29, 1994; and in revised form, December 22, 1994)

Jong-Moon Jeong (§) S. N. Prasanna Murthy James T. Radek Laszlo Lorand (¶)

From the Department of Cell and Molecular Biology and The Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Guinea pig liver transglutaminase (EC 2.3.2.13) displays a Ca-independent binding (K= 10^7M) to the same gelatin-binding domain of human plasma fibronectin that is known to form a very tight complex with the human red cell enzyme. The fibronectin-combining site of the liver transglutaminase was investigated by testing fragments obtained from the parent protein by controlled digestion with endoproteinase Lys-C. Overlay assays, probed with anti-fibronectin antibody, revealed that the fibronectin binding ability of the transglutaminase was encoded in a linear sequence in its 28-kDa N-terminal domain. Removal of the first 7 residues by further digestion of the purified 28-kDa material with endoproteinase Glu-C generated a 27-kDa fragment that, however, showed no binding activity. Thus, residues 1-7 in the liver enzyme seem to be of particular importance for influencing its ability to bind to fibronectin.


INTRODUCTION

Binding of intracellular transglutaminases, upon discharge into the blood circulation, may be one of the most important physiological functions of plasma fibronectin. Association of the human red cell transglutaminase with human plasma fibronectin is virtually instantaneous and extremely tight, involving the gelatin (collagen)-binding regions in the constituent chains of the fibronectin molecule, while still allowing ternary complex formation with gelatin at these sites(1, 2, 3) . A 42-kDa fragment (I^6-II^1-II^2-I^7-I^8-I^9), obtained from fibronectin by thermolytic digestion and purified by gelatin-affinity chromatography, displays as good a binding affinity for the transglutaminase as do the parent chains of fibronectin themselves(4) . Reduction and alkylation of the 42-kDa fragment, however, destroy its ability to associate with the transglutaminase, (^1)suggesting that some features of its tertiary structure are necessary for binding the enzyme.

Inasmuch as the binding occurs in the absence of Ca, it seems that neither a putative conformational change induced in the transglutaminase by the uptake of Ca leading to the unmasking of its active center nor its transamidase activity as such are essential with regard to forming a complex with fibronectin.

Further insight into the fibronectin-binding domains of transglutaminases was sought by examining defined proteolytic fragments of the transglutaminase from guinea pig liver for which primary sequence data are available(5) . Binding activities of the fragments were assayed by overlays with fibronectin and probed with anti-fibronectin antibody. Liver transglutaminase is known to display a Ca-independent binding to human plasma fibronectin, although its affinity is somewhat lower than that of the homospecific red cell enzyme(3) . Nevertheless, as presented in this paper, the association of guinea pig liver transglutaminase with the 42-kDa gelatin-binding domain of human fibronectin is sufficiently tight, so that our findings with fragments of the liver enzyme probably represent essential features of the fibronectin-combining sites in several transglutaminases.


MATERIALS AND METHODS

Transglutaminase was isolated from guinea pig livers (260 g, purchased from Pel-Freez Biologicals, Rogers, AR, homogenized in 520 ml of a pH 7.5 buffer containing 25 mM Tris-HCl, 140 mM NaCl, and 2 mM EDTA with four 30-sec bursts in Waring blender at 4 °C). The homogenate was centrifuged (47,000 times g, Beckman JA18 rotor, 45 min, 4 °C), and the supernatant was passed through 10 layers of cheesecloth. The filtrate (700 ml) was mixed and allowed to bind with occasional stirring for 1 h at 4 °C to DEAE cellulose (400 ml of DE52, Whatman, previously equilibrated with 50 mM Tris-HCl, 1 mM EDTA, pH 7.5). The resin was then washed on a Buchner funnel in succession with a 200-ml solution of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 10 units/ml of Trasylol (FBA Pharmaceuticals, West Haven, CT), with 1 liter of 0.8 strength of the homogenization buffer, and, finally, with 250 ml of the full strength buffer. Proteins bound to the DE52 were eluted with 500 ml of 50 mM Tris-HCl, 250 mM NaCl, 2 mM EDTA, pH 7.5. This eluate (550 ml) was diluted with an equal volume of water and was mixed with 100 ml of DE52 pre-equilibrated as before. The slurry was poured into a column (2.5 times 21 cm), washed with 700 ml of homogenization buffer, and then eluted with 50 mM Tris-HCl, 2 mM dithiothreitol, 2 mM EDTA, 500 mM NaCl, pH 7.0. The eluate (50 ml) was divided into 5 equal aliquots, and CaCl(2) was added to these to final concentrations of 7 mM. Each Ca-containing eluate was passed through an Affi-Gel 15-casein column (2.5 times 10 cm; see (6) ) followed by washing with 120 ml of 50 mM imidazole HCl, 2 mM dithiothreitol, 500 mM NaCl, 6 mM CaCl(2), pH 7.0. Transglutaminase was eluted with 50 mM imidazole HCl, 2 mM dithiothreitol, 2 mM EDTA, 500 mM NaCl, pH 7.0. The collected fractions (5 ml each) were immediately mixed with 150 µl of 200 mM EDTA, and those containing enzyme activity (7) were pooled, concentrated on Centriprep-30 (Amicon Corp., Lexington, MA), and dialyzed against 50 mM Tris-HCl, 1 mM EDTA, pH 7.5 at 4 °C. Human plasma fibronectin was obtained as a by-product of Factor XIII preparation(8) . Protein concentrations were computed by measuring absorbance at 280 nm using an extinction coefficient () of 15.8 for guinea pig liver transglutaminase (9) and 12.8 for human plasma fibronectin(10) .

Binding Studies

The 42-kDa gelatin-binding fragment of human plasma fibronectin (11) was kindly provided by Dr. K. C. Ingham (Holland Laboratory, American Red Cross, Rockville, MD). This protein was reacted with fluorescein isothiocyanate for 75 min under the conditions previously employed (4) to generate the fluorescein-labeled 42K^F material. Based on a molar extinction coefficient of 3.4 times 10^4 for fluorescein at 490 nm(12) , 2 mol of this label were incorporated per mole of the 42-kDa protein fragment. Concentrations of the latter were determined by the Lowry method (13) with bovine serum albumin as a standard.

Fluorescence polarization experiments were performed in a SLM 8000C double-emission spectrofluorometer (SLM-AMINCO, Urbana, IL) with = 490 µm and = 530 nm as described previously(4) .

Proteolytic Digestion of Transglutaminase and Purification of Fragments

A stock solution of transglutaminase (0.4 mg/ml) in 100 mM NH(4)HCO(3), 0.1% SDS, pH 7.8, was digested at 30 °C with endoproteinase Lys-C (Boehringer Mannheim, Indianapolis, IN) as described in the figure legends. Following digestion, samples were incubated with 4% SDS and 150 mM dithiothreitol at 75 °C for 5 min and analyzed by SDS-PAGE (^2)(4.8% stacking/16% resolving acrylamide gel; Laemmli procedure(14) ). Following SDS-PAGE and staining (stained in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol, 10% acetic acid for 10 min; destained in 5% methanol, 10% acetic acid, 2 times 10 min; and rinsed in water), gel bands were cut out and soaked in 100 mM ammonium bicarbonate, 0.1% SDS for 30 min. Protein fragments were then eluted with 50 mM ammonium bicarbonate, 0.1% SDS, pH 7.8, into a membrane cap (molecular mass cutoff, 12 kDa) at 12 mA/glass tube for 5 h using a model 422 Electro-eluter (Bio-Rad). Concentrations of the transglutaminase fragments were measured by the modified Lowry procedure(15) .

The 28-kDa fragment of transglutaminase (0.23 mg/ml in 50 mM ammonium bicarbonate, 0.1% SDS) was further digested with [l/l5] of its weight of endoproteinase Glu-C (Boehringer Mannheim) at 37 °C.

Overlay Assay with Human Plasma Fibronectin

Following SDS-PAGE, proteolytic fragments were transferred to nitrocellulose (BA83, 0.2-µm pore size; Schleicher & Schuell) with a LKB Transphor electroblotting unit (Bromma, Sweden) using 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3, for 2 h at 100 V and 4 °C(16) . Amido black (0.2% naphthol blue black in 10% acetic acid, 50% methanol) was used for staining protein bands on nitrocellulose. For overlay assays, the nitrocellulose was blocked with three changes (10 min each) of 0.05% Tween 20 in 10 mM sodium phosphate-buffered saline and was then immersed for 1 h at room temperature in 80 ml of TPBS containing 200-240 µg of human plasma fibronectin. Control sheets were incubated in TPBS alone. The blots were washed with TPBS (3 times 10 min), incubated overnight in a 1:5000 dilution of polyclonal rabbit antibody (IgG fraction) to human fibronectin (Cappel, Westchester, PA) in TPBS, washed again in TPBS (3 times 10 min), and then treated for 2 h with goat anti-rabbit IgG-alkaline phosphatase conjugate (Promega, Madison, WI) in a 1:5000 dilution of TPBS. After three 10-min washes with TPBS, color was developed in 0.37 mM 5-bromo-3-chloro-3-indolyl phosphate, 0.39 mM nitroblue tetrazolium, 100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl(2), pH 9.5, and the blots were washed in distilled water.

Amino Acid Sequencing

Transglutaminase fragments separated by SDS-PAGE were transferred (1 h at 300 mA at 10 °C, using 10 mM CAPS, pH 11, and 10% methanol) to Immobilon-P polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was then rinsed with water, stained with 0.1% Coomassie Brilliant Blue R-250 in 50% methanol for 5 min, destained with several changes of 50% methanol, 10% acetic acid, and, finally, rinsed with 10% methanol. Protein bands were excised from the air-dried membrane and were sequenced in an Applied Biosystems 177A protein sequencer at the Northwestern University Biotechnology Facility.


RESULTS AND DISCUSSION

It was previously shown (4) that a 42-kDa fragment from the gelatin (collagen)-binding region of human plasma fibronectin molecule can form a very tight complex with human red cell transglutaminase. We now find that the same domain of the fibronectin molecule can also bind the transglutaminase isolated from guinea pig liver, albeit less firmly than the homologous red cell enzyme. The 42-kDa fragment of fibronectin was obtained by thermolytic digestion and was purified by gelatin-affinity chromatography(11) . After labeling with fluorescein, changes in the fluorescence anisotropy (Deltar) of a solution of this tagged protein fragment (42K^F; 41 nM in 75 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.5 mM EDTA at 37 °C) were measured upon incremental additions of purified guinea pig liver transglutaminase (LTG) (up to a concentration of 235 nM). Results of these titrations are shown in Fig. 1, where the lower right-hand inset gives the double-reciprocal relationship between the concentration of LTG in the cuvette (abscissa) and the corresponding change in anisotropy (ordinate). From the extrapolated intercept with the ordinate, the maximal fluorescence anisotropic change of the system could be calculated for an infinite concentration of LTG. This value (Deltar(max) = 0.023) is indicated by the horizontal broken lines in the main graph and also in the upper left-hand inset of Fig. 1. Based on a 1:1 association of LTG with 42K^F, the concentration of free LTG prevailing with each addition of the titrant could be calculated. Measured Deltar values (ordinate) as a function of LTG (abscissa) are presented with a linear scale in the upper left-hand inset and with a logarithmic scale in the main graph as a Klotz plot (17) in Fig. 1. From this analysis, with experimental points extending well beyond half-saturation of 42K^F by LTG, a K(a) = 10^7M was calculated for the 42K^F + LTG &lrhar2; 42K^F:LTG equilibrium.


Figure 1: Changes in fluorescence anisotropy (Deltar; ordinate) of the fluorescein-tagged 42-kDa gelatin-binding fragment from human plasma fibronectin upon admixture of liver transglutaminase (LTG; abscissa). In the lower right-hand inset, titration results are presented in the form of a double-reciprocal plot from which a Deltar(max) = 0.023 was calculated for infinite concentration of LTG. The upper left-handinset gives the relationship between Deltar and [LTG], whereas the main graph shows this with a logarithmic scale of [LTG]. The brokenhorizontallines indicate the calculated value for Deltar(max).



The association of LTG with a specific region of plasma fibronectin prompted us to investigate the relevant binding domain in the LTG ligand. Inasmuch as the primary sequence of LTG was known(5) , proteolytic fragmentation could be carried out in a rather predictable manner. LTG fragments, separated by SDS-PAGE and transferred to nitrocellulose, were overlaid with purified human plasma fibronectin and were immunoblotted with a fibronectin-specific antibody.

Digestion of LTG by endoproteinase Lys-C proved to be a suitable approach for defining linear sequences in this enzyme, which exhibited good fibronectin binding properties (Fig. 2). Treatment of the approximately 77-kDa parent protein by endoproteinase Lys-C progressively gave rise to fragments that displayed such activity with apparent masses of 70, 47, 40, and 28 kDa. In the limit digest with this protease (lane 5 in the top panel, lane 8 in the bottom panel of Fig. 2), only the 40- and 28-kDa products were the main fragments of interest, reacting positively with fibronectin. By contrast, the Coomassie Blue-stained prominent peptide doublet (19-kDa, lane 5 in A or lane 8 in A`) did not bind fibronectin. These three, i.e. 40-, 28-, and 19-kDa LTG fragments, were isolated by electroelution and concentrated. Overlay experiments confirmed that the purified 40- and 28-kDa fragments could bind fibronectin, whereas the 19-kDa fragment could not (data not shown).


Figure 2: Binding of human plasma fibronectin to proteolytic fragments of transglutaminase probed, after SDS-PAGE and transblotting to nitrocellulose, with an overlay assay using anti-fibronectin antibody. Top, purified guinea pig liver transglutaminase (lane 2; 0.385 mg/ml) was digested at 30 °C with endoproteinase Lys-C (3.75 µg/ml) for 20 min (lane 3) and 80 min (lane 4) when the concentration of endoproteinase Lys-C was raised (to 10 µg/ml) for another 13 h 40 min of digestion (lane 5). Lanes 3-5 represent protein samples of 12 µg each. Molecular mass markers (rabbit muscle phosphorylase b, bovine serum albumin, hen egg white ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor, and hen egg white lysozyme, which correspond to molecular masses of 97.4, 66.2, 45, 31, 21.5, and 14.4 kDa) are given in lane1, whereas lane2 represents the parent protein sample (5 µg) of guinea pig liver transglutaminase. The nitrocellulose blot was stained with Amido Black in A. Patterns of overlay assays with human plasma fibronectin are shown in B. As controls, omission of the fibronectin ligand gave no staining in immunoblots with anti-fibronectin antibody in C. Molecular masses for the two major fibronectin-binding fragments of transglutaminase (40 and 28 kDa) are indicated on the right. Bottom, transglutaminase (0.4 mg/ml) was digested at 30 °C with endoproteinase Lys-C (20 µg/ml) for 19, 40, 100, 200, 360, and 660 min (lanes 3-8, corresponding to 12-µg protein samples). Lane2 represents the parent protein (5 µg) before digestion. Lane1 shows molecular mass markers, as above. A gives the Amido Black-stained fragmentation pattern of transglutaminase, and B illustrates the preferential binding of fibronectin in the overlay assay to select fragments of the protein.



The purified 28-kDa fragment was further digested by endoproteinase Glu-C (Fig. 3), which caused a time-dependent degradation of this starting material into several smaller peptides of which two, a 27- and a 14.5-kDa species, were selected for sequence analysis. Although the observation that the 28-kDa starting material could not be sequenced by the Edman procedure (data not shown) indicated that this fragment may have contained the blocked N terminus of LTG(5) , it was essential to confirm this supposition by sequencing some of the endoproteinase Glu-C-derived fragments of 28-kDa. The results of amino acid sequencing (Fig. 4) proved convincingly that the 27-kDa peptide represented a fragment of LTG starting with Arg-8 from the suggested cleavage of the Glu-7-Arg-8 bond (5) and that the 14.5-kDa peptide was obtained from a portion of LTG starting with Ala-70, from a postulated cleavage of the Glu-69-Ala-70 bond(5) . It is thus reasonable to infer that the fibronectin-binding 28-kDa fragment represents the N-terminal domain of LTG from residue 1 to Lys-264 or Lys-272(5) .


Figure 3: Profile of the further digestion of the fibronectin-binding 28-kDa fragment of transglutaminase by endoproteinase Glu-C. Digestion of transglutaminase (0.23 mg/ml; lane2, corresponding to 5 µg of this protein) with endoproteinase Glu-C (15 µg/ml) was carried out at 37 °C for 10, 20, 40, 60, 90, 180, and 300 min (lanes 3-8, each representing 15 µg of protein material). Following separation by SDS-PAGE (4.8% stacking/16% running acrylamide gel) and transfer to polyvinylidene difluoride membrane, color was developed by staining with Coomassie Brilliant Blue. Molecular mass references (marked in kDa) are shown in lane1. The 27- and 14.5-kDa fragments from lane9 were cut out for amino acid sequencing.




Figure 4: Alignments of amino acid sequences from the 27- and 14.5-kDa peptides with the known cDNA-derived sequence and numbering (5) in transglutaminase. The peptides were obtained from the further degradation of the 28-kDa fibronectin-binding fragment of transglutaminase by endoproteinase Glu-C, as shown in Fig. 3.



The electroeluted and concentrated 27-kDa fragment tested negatively in the fibronectin binding overlay assay (Fig. 5), suggesting that the N-terminal primary sequence of LTG upstream from Arg-8 may play a crucial role in the interaction with fibronectin. Amino acid sequences in the N-terminal domains of transglutaminases (Fig. 6A) show a high degree of identity only for a subgroup of the enzymes. Perhaps these are the intracellular transglutaminases that, when discharged into the bloodstream, could form complexes with fibronectin. The recombinant A subunit of human placental Factor XIII (Fig. 6A, HFXIIIa; (18) ) tested negatively in our overlay assay with fibronectin; by contrast, the chicken red blood cell transglutaminase (Fig. 6A, CRBCTgase; (19) ) gave a positive response only with chicken plasma fibronectin as the ligand and not with human plasma fibronectin. (^3)Perhaps that extra N-terminal extension in the chicken red blood cell transglutaminase hinders binding to the human fibronectin. The primary sequence of human red cell transglutaminase is not yet available, but it seems to share sequence homologies with other transglutaminases(23) , which may include the immediate N-terminal sequence as well. As depicted in Fig. 6B, highlighting the functional domains in the linear sequence of guinea pig liver transglutaminase(5, 24) , the fibronectin binding ability of the enzyme is encoded in its 28-kDa N-terminal sequence, and, as discussed above, residues 1-7 may be of particular importance in this regard. Future work might possibly decide whether peptides corresponding to this portion of transglutaminase would mimic this effect of the protein.


Figure 5: A 27-kDa fragment, obtained from the 28-kDa fibronectin-binding domain of transglutaminase, tested negatively in the overlay assay. The purified 28-kDa material (0.2 mg/ml) was further digested (37 °C, 50 mM ammonium bicarbonate, 0.1% SDS; 40 min) with endoproteinase Glu-C (15 µg/ml), and the 27-kDa product was separated by electroelution. SDS-PAGE (4.8% stacking/16% running acrylamide gel) and transblotting to nitrocellulose was performed on a 3-µg sample/lane. A shows staining with Amido Black. B and C are the overlays with and without the fibronectin ligand, respectively, developed by reaction with anti-fibronectin antibody. Lane1, reference standards marked in kDa. Lane3 shows the 27-kDa fragment juxtaposed to the two fibronectin-binding products purified from the earlier fragmentation of transglutaminase by endoproteinase Lys-C, i.e. 40 kDa (lane2) and 28 kDa (lane4).




Figure 6: A, comparison of the amino acid sequence at the N terminus of guinea pig liver transglutaminase (GPLTgase; (5) ) with sequences in other members of this family of enzymes. These include the human endothelial (HETgase; (20) ), mouse macrophage (MMTgase; (20) ), chicken red blood cell (CRBCTgase; (19) ), human keratinocyte (HKTgase; (21) ), and rat keratinocyte (RKTgase; (21) ) transglutaminases and the Factor XIII A protein of human placenta (HFXIIIa; (22) ). B, functional domains of guinea pig liver transglutaminase.




FOOTNOTES

*
This work was aided by Grants HL45168 and HL02212 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biology, College of Natural Sciences, University of Suwon, San 2-2, Wawoo-Ri, Bongdam-Myon, Hwasung-Gun, Kyonggi-Do, Korea 445-743.

To whom correspondence should be addressed: Tel.: 312-503-0591; Fax: 312-503-0590.

(^1)
S. N. P. Murthy and L. Lorand, unpublished results.

(^2)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; TPBS, 0.05% Tween 20 in 10 mM sodium phosphate-buffered saline; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; LTG, guinea pig liver transglutaminase; 42K^F, fluorescein-labeled 42-kDa gelatin-binding fragment of fibronectin.

(^3)
J. E. Dailey and L. Lorand, unpublished results.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.