The family of LAMP-2 proteins arises by alternative splicing from a single gene: characterization of the avian LAMP-2 gene and identification of mammalian homologs of LAMP-2b and LAMP-2c.

The two lysosome-associated membrane proteins, LAMP-1 and LAMP-2, are major integral membrane proteins of the lysosomes. They also occur in the plasma membrane, where they have been discovered independently as principal lactosaminoglycan-bearing glycoproteins and as tumor antigens. Avian LAMP-2 has recently been shown to be encoded by at least three transcripts resulting in variant transmembrane and cytoplasmic domains (Hatem et al., 1995). We report isolation and characterization of chicken genomic clones indicating that the three transcripts are the result of alternative splicing of a single LAMP-2 gene. Only a single LAMP-2, homologous to chicken LAMP-2a, has been described in mammals. To ascertain whether multiple forms of LAMP-2 also occur in mammals, we cloned cDNAs encoding LAMP-2 variants homologous to avian LAMP-2b and LAMP-2c from mouse brain cDNA libraries. Thus, the family of LAMP-2 proteins is conserved from bird to mammals and the diversity is generated by alternative splicing of a single LAMP-2 gene.

Multiple mRNAs encode the avian lysosomal membrane protein LAMP-2, resulting in alternative transmembrane and cytoplasmic domains.

Lysosomal membranes are enriched in extensively glycosylated transmembrane proteins, LAMP-1 and LAMP-2. LAMP-1 proteins have been characterized from several mammalian species and from chickens, but no non-mammalian homologues of LAMP-2 have been described, and no splice variants of either protein have been reported. Here we report the characterization of three cDNA clones encoding chicken LAMP-2. The nucleotide sequences of the cDNAs diverge at their 3' ends within the open reading frame, resulting in sequences that code for three different transmembrane and cytoplasmic domains. Southern analysis suggests that a single gene encodes the common region of chicken LAMP-2. The position of the divergence and the identity of the common sequence are consistent with alternative splicing of 3' exons. Analysis of the mRNAs present in adult chicken tissues suggests tissue-specific expression of the three chicken LAMP-2 variants, with LAMP-2b expressed primarily in the brain. The cytoplasmic domain of LAMP-type proteins contains the targeting signal for directing these molecules to the lysosome. Using chimeras consisting of the lumenal domain of chicken LEP100 (a LAMP-1) and the transmembrane and cytoplasmic domains of the LAMP-2 variants, we demonstrate in transfected mouse L cells that all three LAMP-2 carboxyl-terminal regions are capable of targeting the chimeric proteins to lysosomes. Levels of expression, subcellular distribution, and glycosylation of the LAMP proteins have all been shown to change with differentiation in mammalian cells and to be correlated with metastatic potential in certain tumor cell lines. Alternative splicing of the LAMP-2 transcript may play a role in these changes.

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins.

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

Ca(2+)-regulated serine protease associated with the nuclear scaffold.

The nuclear scaffold (NS) is a proteinaceous network of orthogonally arrayed intermediate filament proteins, termed lamins, which is responsible for nuclear structure. Recent work has demonstrated that a subset of lamins A/C is proteolytically cleaved to produce an ATP-binding protein. This proteolytic cleavage is accomplished by a NS protease activity, which shows a considerable selectivity for lamins A/C and is stringently regulated by Ca2+ in vitro, suggesting that it might also participate in control of NS breakdown in various scenarios. Here, we identify the major NS protease as a novel serine protease with a predominantly chymotryptic-like substrate preference, and we show that even transient perturbations in cytosolic Ca2+ have significant effects on the NS protease activity. This NS protease activity shows extensive similarities to the multicatalytic proteinase complex. In addition to a potential role in control of NS breakdown at mitosis and/or under pathological conditions, this NS protease is also strategically located for other functions, such as inactivation of various oncogenic proteins or maturation-promoting factor.

Alterations in nuclear scaffold constituents during carbon tetrachloride-induced liver regeneration.

Liver regeneration was induced in rats by treatment with CCl4, which results in substantial regenerative activity with a sharp mitotic response 2 days after intoxication. Closely paralleling the mitotic index, we observed fourfold increases in nuclear scaffold nucleoside triphosphatase, an activity thought to participate in nucleocytoplasmic RNA transport and in the 46 kD putative enzyme and its selective photolabeling. Because previous work has indicated that the 46 kD protein may be proteolytically derived from lamins A/C by cleavage at a tyrosine residue at aa376, we investigated the response of lamin A/C transcripts during this regeneration. Surprisingly, Northern blot analyses after CCl4 administration showed low levels of lamin A/C transcripts (which appeared to be predominantly poly[A]-), and we found a decrease in immunoprecipitable lamins A/C from in vitro translation of poly(A)- selected RNA. To circumvent potential problems with such analyses, we used reverse transcription/polymerase chain reaction amplification of lamin A/C transcripts from total cytoplasmic RNA. These assays showed a transient, comparatively minor increase in lamin A/C transcripts 1 day after treatment, but levels rapidly declined from 1 to 3 days and were decreased at 3 to 5 days. However, nuclear scaffold protease activity, which shows a considerable selectivity for lamins A/C and may be involved in derivation of the 46 kD protein, increased in parallel to the mitotic response and increases in nucleoside triphosphatase, as assessed using a nonspecific (Azocoll) protease assay. Assays with a specific tyrosine-containing substrate (Z-Y-Sbenzyl) showed an increase that mirrored that observed with the nonspecific substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Demethylation of ribosomal RNA by hepatocyte microsomal preparations.

Previous work has shown that hypomethylation of rRNA is an important control for protein synthesis in rat hepatocytes, and that the net hypomethylation appears to arise from cytoplasmic events. Here we show that demethylation of rRNA is catalyzed by microsomal preparations. The rRNA demethylation is dependent upon NADPH and is almost completely inhibited by carbon monoxide. Demethylation appears to increase following galactosamine intoxication, a hepatotoxin which induces hypomethylation of rRNA and inhibition of protein synthesis.

The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C.

Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)