THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 19% by The American Society of Biological Chemists, Inc

Vol. 261, No. 31, Issue of November 5, pp. 14833-14836,1986 Printed in U.S.A.

Localization of a Liver Transglutaminase and a Large Molecular Weight Transglutaminase Substrate to a Distinct Plasma Membrane Domain*

(Received for publication, March 26, 1986)

David J. Tyrrellz, Winfield S. Sales, and Charles W. SlifeSlI From the Departments of $Biochemistry and §Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322

Rat liver plasma membranes contain transglutamin- ase activity and a large molecular weight protein ag- gregate that serves as a substrate for this enzyme (Slife, C. W., Dorsett, M. D., Bouquett, G. T., Register, A., Taylor, E., and Conroy, S. (1985) Arch. Biochem. Biophys. 241, 329-336; Slife, C. W., Dorsett, M. D., and Tillotson, M. L. (1986) J. Biol. Chem. 261, 3451- 3456). When purified plasma membranes were soni- cated and the different plasma membrane domains were separated by sedimentation through a linear su- crose gradient, virtually all of the transglutaminase activity and the large molecular weight transglutamin- ase substrate were associated with membrane frag- ments which migrated to a very dense region of the gradient (1.18 g/cm3). The bile canalicular markers, 5”nucleotidase and HA-4 antigen, were predomi- nantly found at 1.11 g/cm3, while most of the sinusoi- dal/lateral marker, CE-9 antigen, was detected at 1.14 g/cm3. Smooth membrane vesicles were observed chiefly at the lighter densities upon morphological analysis, while many filament-bearing, plasma mem- brane segments and junctional complexes were con- tained in the heavy transglutaminase fractions. These data show that the plasma membrane transglutaminase and the large molecular weight transglutaminase sub- strate are associated with a distinct region of the plasma membrane.

Transglutaminases catalyze the covalent cross-linking of proteins by forming t(y-glutamy1)lysine isopeptide linkages (1, 2). The enzymes have been identified in a number of tissues and in many different cell types, but in only a few cases are the biological roles of the enzymes understood. During hemostasis, Factor XIII, a transglutaminase found in the blood, stabilizes the fibrin clot by cross-linking the aggre- gated fibrin molecules (3). As epidermal cells undergo termi- nal differentiation, a variety of soluble and membrane-bound proteins are cross-linked forming the cornified envelope, a chemically resistant structure at the skin surface (4-6). In rats, guinea pigs, and several other mammalian species, a copulatory plug is formed in the vagina following coitus (7). The plug, which contains proteins secreted from seminal

* This work was supported by United States Public Health Service Grants AM 32397 (to C. W. S.) and HD 20497 and HD 00553 ( t o W. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll To whom correspondence and reprint requests should be ad- dressed.

vesicles, is cross-linked by a transglutaminase derived from the prostate gland (8).

In mammalian liver, transglutaminase activity is found in both the soluble and membrane fractions after homogeniza- tion (9-11). The soluble enzyme has been isolated (12, 13) and studied extensively (1, 3), but the biological role of the enzyme remains unknown. Recently, we determined that liver plasma membrane contains nearly all of the membrane-as- sociated transglutaminase activity ( l l ) , as well as proteins which can serve as substrates for the enzyme (14). These proteins, which are part of a large molecular weight aggregate that does not enter an SDS’-polyacrylamide gel upon electro- phoresis, may be covalently cross-linked through c(y-gluta- my1)lysine isopeptide linkages (2, 4, 15, 16). The complex is similar to a proteinaceous material we previously isolated from the plasma membrane which stimulates hepatocyte cell- cell adhesion (17).

This paper reports that the plasma membrane transgluta- minase and the large molecular weight transglutaminase sub- strate are associated with a distinct subpopulation of plasma membrane fragments. The transglutaminase-containing frag- ments migrate to a very dense region of a sucrose gradient, a region which contains intercellular junctional and filament- bearing membrane structures. The transglutaminase and its large molecular weight substrate may be associated with these structures. A preliminary account of this work has been presented (18).

MATERIALS AND METHODS AND RESULTS~

DISCUSSION

Hubbard and her colleagues (20, 26) showed that when purified rat liver plasma membranes were sonicated, the ex- tended membrane sheets were disrupted and a heterogeneous population of vesicles and filament-bearing linear fragments was formed. The different vesicles and fragments, which were derived from distinct regions of the plasma membrane, could be separated by sedimentation through a sucrose density gradient. Using this procedure, we found that the liver plasma membrane transglutaminase was associated with membranes

The abbreviations used are: SDS, sodium dodecyl sulfate; BSA, bovine serum albumin.

Portions of this paper (including “Materials and Methods,” “Re- sults,” and Figs. 1-7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biolog- ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-965, cite the authors, and include a check or money order for $6.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

14833

14834 Liver Plasma Membrane Transglutaminase

that migrated to a very high density (1.18 g/cm3) in the gradient. Only small amounts of the bile canalicular mem- brane markers, 5’-nucleotidase and HA-4 antigen, and the sinusoidal/lateral membrane marker, CE-9 antigen, were ob- served at this density. Using immunoelectron microscopy, Hubbard’s group also showed that the HA-4 and CE-9 anti- gens were uniformly distributed throughout the bile canali- cular and sinusoidal/lateral membranes, respectively (20, 26). Therefore, it appears that the liver plasma membrane trans- glutaminase is associated with a specific plasma membrane domain.

Radiolabeled putrescine was incorporated into proteins in purified plasma membrane sheets by a transglutaminase- mediated reaction (14). When the radioactive membranes were sonicated and the membrane fragments were separated on sucrose gradients, the radioactive profile in the gradients correlated with the transglutaminase activity profile. This indicates that only the transglutaminase-containing, plasma membrane fragments contain substrates that are accessible to the enzyme. This also suggests that “in viuo” the enzyme acts only on substrates located in the vicinity of the transglu- taminase-containing plasma membrane domains. When the [3H]p~tre~cine-labeled plasma membrane fragments were subjected to SDS-polyacrylamide gel electrophoresis followed by fluorography, most of the radioactivity was incorporated into proteins that did not enter the stacking gel. Earlier studies suggested either that this large molecular weight ag- gregate is present in the native membrane or that it is rapidly formed during the incubation with putrescine and calcium (14).

We recently suggested that a transglutaminase may partic- ipate in the process of hepatocyte cell-to-cell adhesion by forming covalently cross-linked protein matrices at sites of cell-to-cell contact (14). This suggestion was based on the following observations:

1. Rat liver plasma membranes specifically stimulate the rate of readhesion of single cell suspensions of rat hepatocytes (32).

2. The adhesion stimulatory activity resides in a large molecular weight proteinaceous material that does not enter an SDS-polyacrylamide stacking gel (17). The material con- tains c(y-glutamy1)lysine isopeptide linkages.

3. Rat liver plasma membranes contain transglutaminase activity (11).

4. The plasma membrane-associated transglutaminase spe- cifically incorporates small molecular weight amines into a large molecular weight proteinaceous material associated with the plasma membrane. The material remains at the top of the stacking gel upon SDS-polyacrylamide gel electrophoresis (14).

The studies presented in this manuscript remain consistent with the suggested role of the plasma membrane-associated transglutaminase. An interesting finding from this report centers on the morphological analysis of the membranes in the transglutaminase-containing sucrose gradient fractions. Many intercellular junctions, which include sites of cell-to- cell contact, were associated with the membranes. Therefore, we must consider these structures and the associated cyto- skeletal components as leading candidates for containing the plasma membrane transglutaminase and the large molecular

weight transglutaminase substrate.

Acknowledgments-We would like to thank Dr. Dean Danner for the use of his laser densitometer, L. Fox and R. Thomas for excellent technical assistance, Dr. Peggy R. Girard for assisting with the immunoblots, and Khaleelah Muwwakkil for assisting with the prep- aration of this manuscript. Polyclonal antisera were generously pro- vided by Dr. Ann Hubbard.

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Liver Plasma Membrane Transglutaminase 14835

14836 Liver Plasma Membrane Transglutaminase

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