lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

The 120-kDa lysosomal membrane glycoprotein (lgp120) is an acidic, heavily glycosylated membrane protein enriched in the lysosomal membrane. To determine the basis for its selective transport to and stability in lysosomes, we have investigated the structure of lgp120. By using an oligonucleotide probe corresponding to the amino terminus of rat lgp120, we isolated and characterized cDNA clones containing the entire coding region. The deduced amino acid sequence demonstrates that lgp120 contains a putative signal peptide, 18 sites for N-linked glycosylation, a single membrane-spanning segment, and a short (11 amino acid) cytosolic tail. The sequence suggests a distinct domain organization, with two luminal glycosylated regions separated by a nonglycosylated proline-rich region. Proteolysis in detergent showed that the protein was not intrinsically resistant to exogenous or endogenous proteases. The N-linked oligosaccharides on lgp120, tetraantennary structures with two lactosamine repeats on one of the branches, were not different from those of glycoproteins on the plasma membrane. lgp120 was similar in its domain organization and portions of its amino acid sequence to the avian 100-kDa lysosomal membrane protein LEP100 [Fambrough, D. M., Takeyasu, K., Lippincott-Schwartz, J., Siegel, N. R. & Somerville, D. (1988) J. Cell Biol. 106, 61-67], and to a distinct 110-kDa lysosomal membrane protein (lgp110) that colocalizes with lgp120. The similarities between lysosomal membrane glycoproteins from diverse species, coupled with the fact that at least two distinct lysosomal membrane glycoproteins are expressed in a single species, indicate the existence of a conserved family of glycoproteins enriched in the lysosomal membrane.

The elaborate cytoskeletal matrix underlying the intestinal epithelial cell brush border (BB) is the hallmark of a mature enterocyte. As such, alterations in this structure are potentially useful as markers aiding in the recognition of subtle defects in cell maturation, such as those accompanying dysplasia and neoplasia. For exploration of this hypothesis, the BB components of human ileal and colonic enterocytes have been compared structurally and biochemically with the well-characterized avian BB, and alterations in the BB cytoskeleton in various states of dysplasia and neoplasia have been identified. Ultrastructural analysis of isolated human ileal BBs indicate that the human BB is structurally homologous to BBs isolated from chicken and other mammalian sources. Like other mammalian BBs (eg, from rat) the terminal web cytoskeleton of the human BB is less extensive than that in the avian BB. Immunochemical analysis of isolated human BBs indicates that the major proteins of the avian microvillar actin bundle, villin, fimbrin, and the 110-kd subunit of the 110K-calmodulin complex, are all present in the human BB. The terminal web protein myosin is also present. Unlike the terminal web of the avian BB, which contains a BB-specific isoform of spectrin, TW 260/240, the human BB contains the more widely distributed spectrin isoform, fodrin. In addition, the human BB contains multiple proteins immunoreactive with antibodies to protein 4.1, a spectrin/actin binding protein that is absent from the avian BB. Immunolocalization studies examining the distribution of the BB-specific microvillar protein, villin, in human colonic mucosa indicate that the localization of this protein is disrupted in certain dysplastic and neoplastic states. Thus, both the expression and/or distribution of BB-specific proteins such as villin may be useful markers for defects in the differentiation state of the enterocyte.

The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.

The primary function of the intestinal epithelial cell is the absorption and transport of nutrients and electrolytes from the lumen of the gut to the organism's blood supply. The absorptive membrane on the lumenal surface ofthe cell-the brush border-has been the subject ofintensive physiological and chemical study . Similarly, the exquisite array of actin filments and associated binding proteins that underlies this membrane is among the best-characterized actin-based cytoskeletal structures in nature . Consequently, one would think that the brush border is a system uniquely suited for the study of the cytoplasmic matrix and the integration of cellular functions. Future studies may prove this to be true, but at present, almost nothing is known about how the cytoskeletal apparatus of the brush border participates in absorption, or any other cellular function of the intestinal epithelial cell . Nevertheless, this subject invites speculation. We first will give an overview of the brush border cytoskeleton and then summarize results of several in vitro experiments that have provided a basis for speculation on the function ofthe brush border cytoskeleton in vivo .

One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding protein of 105-110 kdaltons which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. We have treated isolated, membrane-intact brush borders with ATP and obtained solubilization of the 110-kdalton protein, calmodulin (CM), myosin, and lesser amounts of several other cytoskeletal proteins. Electron micrographs of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with "ballooning" of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with calcium and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110-kdalton protein. This result suggests that associated CM is necessary for the ATP-induced solubilization of the 110-kdalton protein. A 110-kdalton protein-CM complex, with 110-kdalton protein: CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110-kdalton protein-CM complex is an irregular, elongated molecule that ranged in size from 5 X 8 nm to 8 X 14 nm, with a Stokes' radius of 6.1 nm. This 110-kdalton protein-CM complex exhibited no Mg++-ATPase activity and no detectable myosin light chain kinase activity. In co-sedimentation assays, the 110-kdalton protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110-kdalton protein were calcium-independent. Negative stains of F-actin and 110-kdalton protein-CM in the absence of ATP showed loosely organized aggregates of actin with the 110-kdalton protein-CM complex coating the surface of the filaments. On the basis of our data, and in agreement with previous calculations (Matsudaira, P.T., and D.R. Burgess, 1979, J. Cell Biol. 83:667-673), we suggest that the lateral bridge of the microvillus is composed of a dimer of the 110-kdalton protein with four associated calmodulins.

We have investigated several structural aspects of the intestinal epithelial brush border from rachitic chicks. At both the light and electron microscope levels, rachitic brush borders are indistinguishable from controls. Although several of the prominent periodic acid-Schiff-positive proteins of the brush border membrane have slightly slower mobilities on sodium dodecyl sulfate/polyacrylamide gels than do corresponding proteins from control brush borders, the major components of the microvillus core, including subunits of 105, 95, and 68 kilodaltons, actin, and calmodulin, are not detectably different. As assayed by a 125 I-labeled calmodulin gel overlay technique, the same calmodulin-binding proteins are present in rachitic and control brush borders. Two proteins, the 105-kilodalton subunit of the microvillus core and an approximately 30-kilodalton membrane protein, bind calmodulin in a calcium-independent manner. Four cytoskeletal proteins (250, 190, 180, and 150 kilodaltons) and one membrane protein (35 kilodaltons) bind calmodulin only in the presence of calcium. Calcium-dependent solation of microvillus core proteins and calcium-dependent phosphorylation of the 20-kilodalton light chain of brush border myosin both occur as in controls. Our results show that rachintic chicks have brush borders that are quite similar to controls with respect to their ultrastructural organization, constituent contractile proteins, and calcium-dependent regulation of contractility and microvillus core structure. Therefore, the decreased absorption of calcium by intestinal epithelial cells in rachitic chicks is probably not due to gross structural or chemical differences in the brush border cytoskeleton.

The function of the microvillous or brush-border surface of the intestinal epithelial cell is presumably to facilitate nutrient and electrolyte absorption by increasing the surface area of its lumenal membrane. Absorption may also be facilitated by brush-border motility of some sort; directly beneath and attached to the brush-border membrane is one of the most extensive and highly organized cytoskeletal apparatuses in nature. Constituent proteins of the brush-border “cytoskeleton” include actin, myosin, and a variety of associated proteins that are subdivided into two structural, and probably functional, domains: the microvillus and the terminal web (for an extensive review of brush-border structure and contractile proteins, see Mooseker and Howe [1981]). Each microvillus contains a bundle of about 20 actin filaments that have uniform polarity. Using terminology based on the arrowhead morphology of heavy meromyosin (HMM) or S1-actin-filament complexes, the “barbed” ends of microvillus core filaments are at the tip end of the...

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle. Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure. The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons.The supernate from the brush-border preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons. The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca++, and (c) 4--5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca++. With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin. In the presence of Ca++ (greater than 5 x 10(-7) M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable from. The dissociation of calmodulin was reversed by removal of Ca++. If microvillus cores were pretreated with phalloidin, the Ca++-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1:10 in the demembranated brush border and 1:2-3 in the microvillus core. No calmodulin was detected in the detergent-solubilized brush-border membrane fraction.