Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein.

Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173-178-residue stretch of the human 37LRP sequence, has been identified as the laminin-1-binding site. Peptides from 37LRP of species that contain this palindrome-bind laminin-1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15-residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin-1-binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin-1-binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin-1-binding affinity of the peptides influences their inhibitory activity.     

The 37‐kDa laminin receptor precursor regulates the malignancy of human glioma cells

Glioma is one of the most common brain tumors and one of the most aggressive cancers. Although extensive progress has been made regarding to the diagnosis and treatment, the mortality in glioma patients is still high. Therefore, finding new therapeutic targets to the glioma is critical to the advancement in cancer treatment. Recently, the 37‐kDa laminin receptor precursor (37LRP) was reported to play important roles in occurrence of some types of cancer, indicating that this molecule may function as a key regulator in the tumor migration and metastasis. However, there is still no report to elucidate the correlation between 37LRP expression and glioma genesis and development. In this study, we found the higher expression of 37LRP in the glioma cells compared with the normal brain cells. We also indicated that the downregulation of 37LRP could affect the glioma biomarker expression and also weaken the proliferative, migratory, and metastatic capacity of glioma cells in vitro. Furthermore, 37LRP silencing inhibited the glioma tumor growth in vivo. Collectively, these data demonstrated that 37LRP regulates the metastasis of glioma cells in vitro and tumor growth in vivo, suggesting that 37LRP may function as a potential molecular target in the glioma treatment.     

Subcellular localization of prion proteins and the 37 kDa/67 kDa laminin receptor fused to fluorescent proteins

The 37 kDa/67 kDa laminin receptor LRP/LR acts as a receptor for both PrPc and PrPSc at the cell surface. Here, we further analyzed the subcellular localization of fluorescent labeled prion protein (PrP) and laminin receptor (LRP/LR) molecules. We show that EGFP-PrP is localized at the cell surface and in a perinuclear compartment, respectively. In contrast, a DsRed-DeltaSP-PrP mutant lacking the signal peptide is almost exclusively found in the nucleus but does not colocalize with heterochromatin. Interestingly, LRP-DsRed efficiently colocalizes with EGFP-PrP in the perinuclear compartment and LRP-ECFP partly colocalizes with DsRed-DeltaSP-PrP in the nucleus, respectively. We conclude that the interactions of PrP and LRP/LR are not restricted to the cell surface but occur also in intracellular compartments suggesting a putative role of LRP/LR in the trafficking of PrP molecules.     

Characterization of the porcine multicopy ribosomal protein SA/37-kDa laminin receptor gene family

Prions represent a new class of infectious agents. The pathogenic prion protein (PrPSc) is known as the trigger of bovine transmissible spongiform encephalopathy (TSE). By contrast, an oral transmission of PrPSc and an ensuing infection seems to be blocked in non-ruminants such as pigs. Several investigations postulate that the ribosomal protein SA (RPSA) previously named 37-kDa laminin receptor precursor (LRP)/67-kDa laminin receptor (LR) is the candidate for binding and internalization of externally added cellular prion protein in the gut. We isolated a porcine ribosomal protein SA cDNA that consists of 1064 bp with an open reading frame of 885 bp encoding a 295 aa protein. The alignment of vertebrate ribosomal protein SA sequences displayed interspecies differences between cattle and pigs at positions 241 and 272 in the putative indirect PrP interaction site (aa 180-285) on RPSA. A PAC library screen revealed the existence of two processed ribosomal protein SA pseudogenes (RPSAP1 and RPSAP3) and of one non-processed pseudogene (RPSAP2). The pseudogenes have been assigned to SSC6 and SSC1 by hybrid panel analyses and FISH. Compared with the porcine cDNA 3, 7, and 13 insdels, 36, 25, and 57 single nucleotide exchanges and 6, 10, and 8 premature stop codons have been deciphered for RPSAP1, RPSAP2, and RPSAP3. In the 5', 3', and intron like regions, 2 (RPSAP1), 10 (RPSAP2), and 4 (RPSAP3) repeats have been detected. Basically, the repeats belong to one of the class/family LINE/L1, SINE/tRNA-Glu and DNA/MER1_type. We conclude that the pig genome contains multiple copies of the RPSA sequence probably as a consequence to maintain the multifunctionality of the mature protein.     

The prion-like protein Doppel fails to interact with itself, the prion protein and the 37 kDa/67 kDa laminin receptor in the yeast two-hybrid system

The prion-like protein termed Doppel (Dpl) shows approx. 25% sequence identity with all known prion proteins (PrP). We recently showed that the cellular PrP is dimeric under native conditions, a finding which was confirmed by the investigation of its crystal structure. Human PrP further interacts with its cellular receptor, the 37 kDa/67 kDa laminin receptor (LRP/LR). Here we report that human Doppel fails to interact with (i). itself, (ii). the human 37 kDa/67 kDa LRP/LR, and (iii). the human cellular prion protein (huPrP) in the yeast two-hybrid system. Our findings suggest that Dpl and PrP are not related or are only marginally related with respect to their ligand binding behaviour.     

Crystal structure of the human laminin receptor precursor

The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.     

Characterization of the human cyclophilin gene and of related processed pseudogenes

The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T-cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Sp1 binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non-coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin-related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes.     

Identification of two processed pseudogenes of the human Tom20 gene

The open reading frame (ORF) of the human Tom20 gene (hTom20) was amplified by PCR from a HeLa cDNA library using primers based on the sequence of HUMRSC145 and cloned into a pET15b vector. Amplification of human genomic DNA using these primers yielded a DNA fragment of the same size as that of the ORF of hTom20 cDNA. Sequencing of this fragment revealed that: (1) it has the same number of base pairs as the ORF of hTom20 cDNA (438 bp); and (2) the two sequences differ by 14 single base pair substitutions (97% similarity) causing eight changes in the amino acid sequence and two premature stop codons. Further amplification of human genomic DNA adaptor-ligated libraries using primers based on HUMRSC145 revealed three different sequence-related genomic regions; one corresponding to the fragment referred above, another corresponding to the hTom20 gene, and a third fragment of which the sequence differs from the ORF of hTom20 cDNA by only 22 base pair substitutions and a deletion of 4 bp. We conclude that, in addition to the hTom20 gene, there are two genomic DNA sequences (Ψ1Tom20 and Ψ2Tom20) that are processed pseudogenes of hTom20. Aspects concerning their evolutionary origin are discussed. Received: 12 September 1997 / Accepted: 29 November 1997     

Interaction of Doppel with the full-length laminin receptor precursor protein

Doppel (Dpl) is a homolog of normal cellular prion protein (PrPc) with unknown functions. Ectopic expression of Dpl in the central nervous system (CNS) causes neurotoxicity and this effect is rescued by the expression of PrPc. However, the molecular basis for the protective effect of PrPc remains unclear. Using a yeast two-hybrid system, we showed that Dpl binds the full-length 37-kDa laminin receptor precursor protein (LRP), one of the receptors of PrPc. The interaction was also validated by immunoprecipitation and immunoblotting using transfected cell lines and in vivo derived tissues. Further mapping experiments showed that although the middle fragment containing residues 100-220 of LRP was able to interact with Dpl, deletion of the N-terminal domain of the full-length LRP abolished its interaction with Dpl. These results suggest that while both PrPc and Dpl interact with LRP, the domains that are involved in the binding are not the same. Our results may have implications for the molecular mechanisms of Dpl-PrPc antagonism and physiological roles of Dpl.     

Truncated carboxyl-terminal fragments of beta-amyloid precursor protein are processed to amyloid beta-proteins 40 and 42

We previously showed that beta-amyloid precursor protein (APP) is cleaved not only in the middle of the membrane (gamma-cleavage) but also at novel cleavage sites close to the membrane/cytoplasmic boundary (epsilon-cleavage), releasing APP intracellular domains (AICDs) 49-99 and 50-99. To learn more about the relationship between gamma- and epsilon-cleavage, C-terminally truncated carboxyl-terminal fragments (CTFs) of APP, especially CTFs1-48 and 1-49 (the postulated products that are generated by epsilon-cleavage), were transiently expressed in CHO cells. Most importantly, the cells expressing CTF1-49 secreted predominantly amyloid beta-protein (Abeta) 40, while those expressing CTF1-48 secreted preferentially Abeta42. This supports our assumption that epsilon-cleavage precedes Alphabeta production and that preceding epsilon-cleavage determines the preference for the final Abeta species. The gamma-secretase inhibitors, L-685,458 and DAPT, suppressed Abeta production from CTF1-49. Regarding Abeta production from CTF1-48, L-685,458 suppressed it, but DAPT failed to do so. A dominant negative mutant of presenilin 1 suppressed the production of Abeta40 and 42 from both CTFs1-48 and 1-49. These data should shed significant light into the mechanism of Abeta production.     

The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein.

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.     

Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family.

The functional gene and three intronless pseudogenes for human triosephosphate isomerase were isolated from a recombinant DNA library and characterized in detail. The functional gene spans 3.5 kilobase pairs and is split into seven exons. Its promoter contains putative TATA and CCAAT boxes and is extremely rich in G and C residues (76%). The pseudogenes share a high degree of homology with the functional gene but contain mutations that preclude the synthesis of an active triosephosphate isomerase enzyme. Sequence divergence calculations indicate that these pseudogenes arose approximately 18 million years ago. We present evidence that there is a single functional gene in the human triosephosphate isomerase gene family.     

Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.     

Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor.

Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP). Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 [amino acids (aa) 144-179] as a direct and PrPLRPbd2 (aa 53-93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP. The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179. Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP. Considering the absence of LRP homodimerization and the direct and indirect LRP-PrP interaction sites, we propose a comprehensive model for the LRP-PrP-HSPG complex.     

Midkine binds to 37-kDa laminin binding protein precursor, leading to nuclear transport of the complex

Midkine (MK) is a heparin binding multifunctional protein that promotes cell survival and cell migration. MK was found to bind to 37-kDa laminin binding protein precursor (LBP), a precursor of 67-kDa laminin receptor, with K(d) of 1.1 nM between MK and LBP-glutathione-S-transferase fusion protein. The binding was inhibited by laminin, anti-LBP, amyloid beta-peptide, and heparin; the latter two are known to bind to MK. In CMT-93 mouse rectal carcinoma cells, LBP was mostly located in the cytoplasm as revealed by immunostaining with anti-LBP antibody. That a portion of LBP or 67-kDa laminin receptor was located at the surface of these cells was verified by inhibition of cell attachment to laminin-coated dishes by anti-LBP antibody. When MK was added to culture medium of these cells, a part of LBP migrated to the nucleus. The movement occurred concomitantly with nuclear transport of biotin-labeled MK. These findings suggested that the binding of MK to LBP caused nuclear translocation of the molecular complex.     

Characterization and chromosome localization of a processed pseudogene related to the bovine laminin receptor gene family

A bovine BAC clone containing a processed laminin receptor pseudogene (LAMR1P) has been isolated and characterized. A 2,901-bp sequence was produced from the clone, of which 1,187 bp represented seven identifiable exon-like domains, but no intervening sequences. The pseudogene sequence reveals several transversions and transitions, as well as insertions and deletions. A premature stop codon motif is present at nucleotide position 115 located in the exon-2-like domain. Physical mapping of the gene was performed by FISH and RH panel mapping and assigned LAMR1P to BTA4q24-->q26 with the closest linkage to BM6458 (19 cR, LOD score of 11.6). The functional laminin receptor putatively plays an important role in the transmission of bovine spongiform encephalopathy (BSE). In this process, the receptor supposedly acts as the binding site for prion proteins to enter mammalian cells. Considering the existence of several human laminin receptor pseudogenes forming a complex family, any knowledge of even pseudogene sequences might be helpful to isolate the functional bovine laminin receptor gene.