Advertisement

Membrane Amine Oxidase Cloning and Identification as a Major Protein in the Adipocyte Plasma Membrane*

  • Nicholas J. Morris
    Affiliations
    Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195
    Search for articles by this author
  • Axel Ducret
    Affiliations
    Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195
    Search for articles by this author
  • Ruedi Aebersold
    Affiliations
    Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195
    Search for articles by this author
  • Stuart A. Ross
    Affiliations
    Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195
    Search for articles by this author
  • Susanna R. Keller
    Affiliations
    Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195
    Search for articles by this author
  • Gustav E. Lienhard
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 and
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant DK 25336 (to G. E. L.), a Juvenile Diabetes Foundation International Fellowship (to N. J. M.), a National Research Service Award (to S. A. R.), and funding (to R. A.) from the National Science Foundation Science and Technology Center for Molecular Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U72632.
Open AccessPublished:April 04, 1997DOI:https://doi.org/10.1074/jbc.272.14.9388
      A 97-kDa protein present in the glucose transporter (GLUT4 isotype)-containing vesicles from rat adipocytes has been isolated, the sequences of two tryptic peptides were obtained, and on the basis of these its cDNA partially cloned. The 97-kDa protein is almost certainly identical to a major integral glycoprotein of this size in the rat adipocyte plasma membrane, since its predicted N-terminal sequence is the same as that recently determined for this glycoprotein by amino acid sequencing. Moreover, the predicted partial sequence (322 amino acids) of the 97-kDa protein is highly homologous to the corresponding region of a human placental amine oxidase, which was cloned simultaneously and proposed to be a secreted protein. The amino acid sequence of the 97-kDa rat/human amine oxidase indicates that the protein consists of a very short N-terminal cytoplasmic domain followed by a single transmembrane segment and a large extracellular domain containing the catalytic site. Thus this study establishes the 97-kDa rat/human amine oxidase as the first integral membrane amine oxidase to be cloned. The membrane amine oxidase was more abundant in the plasma membranes than the low density microsomes of the adipocyte, and in contrast to some other proteins found in GLUT4 vesicles, it did not redistribute to the plasma membrane in response to treatment of the cells with insulin.

      INTRODUCTION

      Primary amine oxidases form a family of enzymes that catalyze the oxidation of primary amines by molecular oxygen, to yield the corresponding aldehyde, ammonia, and hydrogen peroxide. Typically these enzymes consist of two identical subunits; each contains an oxidized tyrosine residue, known as topa quinone, and one atom of copper, both of which participate in catalysis (reviewed in Refs.
      • Klinman J.P.
      • Mu D.
      and
      • Hartmann C.
      • McIntire W.S.
      ). Several secreted members of this family, including bovine serum amine oxidase and kidney diamine oxidase, have been cloned and extensively characterized (
      • Mu D.
      • Medzihradszky K.F.
      • Adams G.W.
      • Mayer P.
      • Hines W.M.
      • Burlingame A.L.
      • Smith A.J.
      • Cai D.
      • Klinman J.P.
      ,
      • Novotny W.F.
      • Chassande O.
      • Baker M.
      • Lazdunski M.
      • Barbry P.
      ). By contrast considerably less is known about one (or possibly more) suspected member of this family. This is the membrane-bound amine oxidase that is highly susceptible to inhibition by semicarbazide, and is often referred to as the “tissue-bound semicarbazide-sensitive” amine oxidase (
      • Lyles G.A.
      ,
      • Callingham B.A.
      • Crosbie A.E.
      • Rous B.A.
      ,
      • Buffoni F.
      ). This enzyme, as detected by its activity against benzylamine, has been found to be present in a large number of tissues and cell types, including vascular smooth muscle cells, white and brown adipocytes, and skin fibroblasts (
      • Lyles G.A.
      ,
      • Callingham B.A.
      • Crosbie A.E.
      • Rous B.A.
      ,
      • Buffoni F.
      ). Its cloning as such (see below) has not been reported, and whether it is also of the topa quinone and copper-containing type has not been established.
      The present study began as part of our investigation of insulin stimulation of glucose transport. Treatment of fat and muscle cells with insulin causes a rapid elevation in glucose transport due to a rapid increase in the amount of the glucose transporter (GLUT4 isotype)
      The abbreviations used are: GLUT4
      glucose transporter isotype 4
      HPAO
      human placental amine oxidase
      RACE
      rapid amplification of cDNA ends
      vp97
      97-kDa protein in GLUT4 vesicles
      PCR
      polymerase chain reaction
      bp
      base pair(s)
      in the plasma membrane. The basis for this increase in amount is largely the enhanced trafficking of GLUT4 from intracellular locations to the plasma membrane (reviewed in
      • Holman G.D.
      • Cushman S.W.
      ). We have developed a procedure for isolating the intracellular membranes containing GLUT4 (referred to as GLUT4 vesicles) from adipocytes and are characterizing other proteins present in these vesicles (
      • Mastick C.C.
      • Aebersold R.
      • Lienhard G.E.
      ). In the course of this work, we partially cloned a 97-kDa vesicle protein. This protein, which we initially referred to as vp97, has proven to be a membrane-bound, semicarbazide-sensitive amine oxidase that is almost certainly identical to a major integral glycoprotein in the adipocyte plasma membrane. As our investigation of this protein was nearing completion, the cloning of its human homolog was reported (
      • Zhang X.
      • McIntire W.S.
      ). However, the human homolog, whose properties as protein were not examined experimentally, was proposed to be another secreted oxidase, rather than a membrane-bound one. Thus, our results identify and characterize the first cloned membrane amine oxidase.

      DISCUSSION

      The combined results from this study, the partial characterization of a major integral glycoprotein in adipocyte plasma membranes (
      • Jochen A.
      • Guven S.
      • Hays J.
      ), and the cloning of HPAO (
      • Zhang X.
      • McIntire W.S.
      ) show that vp97/HPAO is a semicarbazide-sensitive membrane amine oxidase. Moreover, since comparison of the complete amino acid sequence of HPAO with that of other amine oxidases revealed that it is a topa quinone and copper-containing enzyme (
      • Zhang X.
      • McIntire W.S.
      ), the membrane amine oxidase has this type of catalytic domain.
      Raimondi et al. (
      • Raimondi L.
      • Pirsino R.
      • Ignesti G.
      • Capecchi S.
      • Banchelli G.
      • Buffoni F.
      ) originally reported that white adipocytes contained a membrane-bound, semicarbazide-sensitive amine oxidase, for which benzylamine was a good substrate (Km = 12 μM). Our observation that the activity is highest in the plasma membrane agrees with the results of these authors, who found the highest activity in the crude membrane fraction that would be most enriched in plasma membranes (
      • Raimondi L.
      • Pirsino R.
      • Ignesti G.
      • Capecchi S.
      • Banchelli G.
      • Buffoni F.
      ). Moreover, localization of the oxidase in pig adipocytes by immunofluorescence with cross-reacting antibodies raised against the bovine serum oxidase showed the protein to be on the cell surface (
      • Raimondi L.
      • Pirisino R.
      • Banchelli G.
      • Ignesti G.
      • Conforti L.
      • Romanelli E.
      • Buffoni F.
      ).
      We isolated the membrane-amine oxidase as a component of the GLUT4 vesicles. Other components of these vesicles, including GLUT4 itself and the 165-kDa membrane aminopeptidase, translocate to the plasma membrane in response to insulin (
      • Mastick C.C.
      • Aebersold R.
      • Lienhard G.E.
      ). However, as assessed both by staining for the protein and by activity, the membrane amine oxidase did not do so. One likely explanation for the difference derives from the nature of the GLUT4 vesicles isolated by immunoadsorption with anti-GLUT4. A recent study has shown that the vesicles are not homogeneous; a portion are fragmented endosomal membranes, and another portion may be specialized small secretory vesicles (
      • Martin S.
      • Tellam J.
      • Livingstone C.
      • Slot J.W.
      • Gould G.W.
      • James D.E.
      ). Insulin probably increases the rate at which vesicles traffic from the endosomal system to the plasma membrane (
      • Tanner L.I.
      • Lienhard G.E.
      ) and also may increase the rate at which the specialized secretory vesicles fuse with the plasma membrane (
      • Holman G.D.
      • Lo Leggio L.
      • Cushman S.W.
      ), but insulin treatment may not significantly reduce the membrane area of the endosomal system. If the latter is the case, the amount of GLUT4 vesicles derived from fragmented endosomes in insulin-treated cells may be about the same as for untreated cells. Thus if the intracellular portion of the membrane amine oxidase is primarily in endosomes rather than specialized secretory vesicles, then the amount of oxidase found in the GLUT4 vesicles, and the low density microsomes from which they are derived, will not decrease in response to insulin. The finding that the oxidase is more abundant in the plasma membranes than the low density microsomes is consistent with the fact that its short cytoplasmic domain contains neither the tyrosine nor the dileucine motif required for selective endocytosis from the plasma membrane (
      • Sandoval I.V.
      • Baake O.
      ).
      The physiological role of the membrane amine oxidase is not known. It may function to degrade bioactive amines, such as histamine, or amine products of intermediary metabolism, such as methylamine and aminoacetone (
      • Lyles G.A.
      ,
      • Callingham B.A.
      • Crosbie A.E.
      • Rous B.A.
      ,
      • Buffoni F.
      ). These compounds have been shown to be substrates in vitro (
      • Lyles G.A.
      ,
      • Callingham B.A.
      • Crosbie A.E.
      • Rous B.A.
      ,
      • Buffoni F.
      ). The identification and cloning of the membrane amine oxidase should open the way to investigate its role in vivo For example, targeted disruption of its gene in mice is now feasible.

      Acknowledgments

      We are indebted to Dr. Judith Klinman for helpful advice about amine oxidases, and to Dr. William McIntire for both advice and preprints of articles in press. We thank Joshua Sparling for technical assistance and Mary Harrington for expert secretarial assistance.

      REFERENCES

        • Klinman J.P.
        • Mu D.
        Annu. Rev. Biochem. 1994; 63: 299-344
        • Hartmann C.
        • McIntire W.S.
        Methods Enzymol. 1996; (in press)
        • Mu D.
        • Medzihradszky K.F.
        • Adams G.W.
        • Mayer P.
        • Hines W.M.
        • Burlingame A.L.
        • Smith A.J.
        • Cai D.
        • Klinman J.P.
        J. Biol. Chem. 1994; 269: 9926-9932
        • Novotny W.F.
        • Chassande O.
        • Baker M.
        • Lazdunski M.
        • Barbry P.
        J. Biol. Chem. 1994; 269: 9921-9925
        • Lyles G.A.
        Prog. Brain Res. 1995; 106: 293-303
        • Callingham B.A.
        • Crosbie A.E.
        • Rous B.A.
        Prog. Brain Res. 1995; 106: 305-321
        • Buffoni F.
        Prog. Brain Res. 1995; 106: 323-331
        • Holman G.D.
        • Cushman S.W.
        BioEssays. 1994; 16: 753-759
        • Mastick C.C.
        • Aebersold R.
        • Lienhard G.E.
        J. Biol. Chem. 1994; 269: 6089-6092
        • Zhang X.
        • McIntire W.S.
        Gene (Amst.). 1996; 179: 279-286
        • Paz M.A.
        • Flückiger R.
        • Boak A.
        • Kagan H.M.
        • Gallop P.M.
        J. Biol. Chem. 1991; 266: 689-692
        • Wessel D.
        • Flügge U.I.
        Anal. Biochem. 1984; 138: 141-143
        • Ducret A.
        • Foyn Bruun C.
        • Bures E.J.
        • Marhaug G.
        • Husby G.
        • Aebersold R.
        Electrophoresis. 1996; 17: 866-876
        • Simpson I.A.
        • Yver D.R.
        • Hissin P.J.
        • Wardzala L.J.
        • Karnieli E.
        • Salans L.B.
        • Cushman S.W.
        Biochim. Biophys. Acta. 1983; 763: 393-407
        • Altschul S.F.
        • Gish W.
        • Miller W.
        • Myers E.W.
        • Lipman D.J.
        J. Mol. Biol. 1990; 215: 403-410
        • Neumann R.
        • Hevey R.
        • Abeles R.H.
        J. Biol. Chem. 1975; 250: 6362-6367
        • Dalphin M.E.
        • Brown C.M.
        • Stockwell P.A.
        • Tate W.P.
        Nucleic Acids Res. 1996; 24: 216-218
        • Jochen A.
        • Guven S.
        • Hays J.
        Mol. Membr. Biol. 1995; 12: 277-281
        • Claros M.G.
        • von Heijne G.
        Comput. Appl. Biosci. 1994; 10: 685-686
        • Keller S.R.
        • Scott H.M.
        • Mastick C.C.
        • Aebersold R.
        • Lienhard G.E.
        J. Biol. Chem. 1995; 270: 23612-23618
        • Raimondi L.
        • Pirsino R.
        • Ignesti G.
        • Capecchi S.
        • Banchelli G.
        • Buffoni F.
        Biochem. Pharmacol. 1991; 41: 467-470
        • Raimondi L.
        • Pirisino R.
        • Banchelli G.
        • Ignesti G.
        • Conforti L.
        • Romanelli E.
        • Buffoni F.
        Comp. Biochem. Physiol. 1992; 102B: 953-960
        • Martin S.
        • Tellam J.
        • Livingstone C.
        • Slot J.W.
        • Gould G.W.
        • James D.E.
        J. Cell Biol. 1996; 134: 625-635
        • Tanner L.I.
        • Lienhard G.E.
        J. Biol. Chem. 1987; 262: 8975-8980
        • Holman G.D.
        • Lo Leggio L.
        • Cushman S.W.
        J. Biol. Chem. 1994; 269: 17516-17524
        • Sandoval I.V.
        • Baake O.
        Trends Cell Biol. 1994; 4: 292-297