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1.  Laminin-111-derived peptides and cancer 
Cell Adhesion & Migration  2013;7(1):150-159.
Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the β1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for cancer.
doi:10.4161/cam.22827
PMCID: PMC3544779  PMID: 23263633
laminin-111; synthetic peptide; metastasis; tumor growth; angiogenesis; migration; adhesion; basement membrane; proteases
2.  An Antibody to the Lutheran Glycoprotein (Lu) Recognizing the LU4 Blood Type Variant Inhibits Cell Adhesion to Laminin α5 
PLoS ONE  2011;6(8):e23329.
Background
The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM.
Methodology/Principal Findings
In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg175, the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5.
Conclusion/Significance
This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.
doi:10.1371/journal.pone.0023329
PMCID: PMC3155534  PMID: 21858073
3.  Identification of α-Dystroglycan Binding Sequences in the Laminin α2 Chain LG4-5 Module 
The biological activities of the laminin α2 chain LG4-5 module result from interactions with cell surface receptors, such as heparan sulfate proteoglycans and α-dystroglycan. In this study, heparin and α-dystroglycan binding sequences were identified using 42 overlapping synthetic peptides from the LG4-5 module and using recombinant LG4-5 protein (rec-α2LG4-5). Physiological activities of the active peptides were also examined in explants of submandibular glands. Heparin binding screens showed that the A2G78 peptide (GLLFYMARINHA) bound to heparin and prevented its binding to rec-α2LG4-5. Furthermore, alanine substitution of the arginine residue in the A2G78 site on rec-α2LG4-5 decreased heparin binding activity. When α-dystroglycan binding of the peptides was screened, two peptides, A2G78 and A2G80 (VQLRNGFPYFSY), bound α-dystroglycan. A2G78 and A2G80 also inhibited α-dystroglycan binding of rec-α2LG4-5. A2G78 and A2G80 specifically inhibited end bud formation of submandibular glands in culture. These results suggest that the A2G78 and A2G80 sites play functional roles as heparan sulfate- and α-dystroglycan-binding sites in the module. These peptides are useful for elucidating molecular mechanisms of heparan sulfate- and/or α-dystroglycan-mediated biological functions of the laminin α2 chain.
doi:10.1016/j.matbio.2009.09.006
PMCID: PMC2826543  PMID: 19800000
laminin; synthetic peptide; basement membrane; heparin; α-dystroglycan
4.  Chain-Specific Heparin-Binding Sequences in the Laminin α Chain LG45 Modules 
Biochemistry  2009;48(23):5375-5381.
Laminin α chains contain five tandem globular modules (LG1-5) at the C-terminus. Here, we focused on the LG45 module, which play a critical biological role via binding to heparin/heparan sulfate, and examined their chain-specific heparin-binding affinity. The relative heparin-binding affinity of recombinant laminin α chain LG45 proteins was α5> α4> α1> α2 and α3. The α5 chain LG45 module also promoted the strongest cell attachment. We screened heparin-binding sequences using the recombinant α5LG45 protein and 43 synthetic peptides. Four peptides, A5G71 (GPLPSYLQFVGI) (IC50 = 91.8 µM), A5G77 (LVLFLNHGHFVA) (IC50 = 7.0 µM), A5G81 (AGQWHRVSVRWG) (IC50 = 5.9 µM), and A5G94 (KMPYVSLELEMR) (IC50 = 0.84 µM), inhibited the heparin-binding of rec-α5LG45. Additionally, the same four peptides exhibited dose-dependent heparin-binding activity in a solid-phase assay. We found that the α5 chain LG45 module contains four heparin-binding sequences and this number is higher than that of the other LG45 modules (α2 and α3, 1 sequence; α1 and α4, 2 sequences). The data suggest that the active sequences identified from the synthetic peptide screening contribute to the heparin-binding activity of the LG45 module. Most of the heparin-binding sequences in the LG45 modules are located in the N-terminal regions of the LG4 module within the loop regions in the proteins. The data suggest that the N-terminal loop regions of the LG4 module are mainly involved in the heparin/heparan sulfate-mediated biological functions.
doi:10.1021/bi900542u
PMCID: PMC2756285  PMID: 19415899
laminin; basement membrane; heparin; peptide; chain specificity
5.  Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin α5 in the glomerular basement membrane 
The Journal of Cell Biology  2003;161(1):187-196.
In developing glomeruli, laminin α5 replaces laminin α1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin α5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin α5 domains VI through I fused to the human laminin α1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 −/− background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 −/− glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin α5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the α5 G domain essential for mesangial cell adhesion to α5LG3-5. Finally, in vitro studies showed that integrin α3β1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin α5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin α5 in the GBM.
doi:10.1083/jcb.200211121
PMCID: PMC2172883  PMID: 12682087
mesangium; cell adhesion; kidney glomerulus; integrin α3β1; transgenic mice
6.  Opposing Roles of Integrin α6Aβ1 and Dystroglycan in Laminin-mediated Extracellular Signal-regulated Kinase Activation 
Molecular Biology of the Cell  2003;14(5):2088-2103.
Laminin–integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed β1-containing integrins and dystroglycan but lacked integrin α6β4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the α3β1and α6β1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin α6β1 and not by α3β1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin α6β1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin α6 splice variants, α6A and α6B, whereas the nonresponding cell line expressed only α6B. Furthermore, ERK activation was seen in cells transfected with the integrin α6A subunit, but not in α6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin α6Aβ1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.
doi:10.1091/mbc.E03-01-0852
PMCID: PMC165099  PMID: 12802077
7.  Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases 
Journal of Clinical Investigation  2003;111(5):707-716.
Kidney disease affects over 20 million people in the United States alone. Although the causes of renal failure are diverse, the glomerular filtration barrier is often the target of injury. Dysregulation of VEGF expression within the glomerulus has been demonstrated in a wide range of primary and acquired renal diseases, although the significance of these changes is unknown. In the glomerulus, VEGF-A is highly expressed in podocytes that make up a major portion of the barrier between the blood and urinary spaces. In this paper, we show that glomerular-selective deletion or overexpression of VEGF-A leads to glomerular disease in mice. Podocyte-specific heterozygosity for VEGF-A resulted in renal disease by 2.5 weeks of age, characterized by proteinuria and endotheliosis, the renal lesion seen in preeclampsia. Homozygous deletion of VEGF-A in glomeruli resulted in perinatal lethality. Mutant kidneys failed to develop a filtration barrier due to defects in endothelial cell migration, differentiation, and survival. In contrast, podocyte-specific overexpression of the VEGF-164 isoform led to a striking collapsing glomerulopathy, the lesion seen in HIV-associated nephropathy. Our data demonstrate that tight regulation of VEGF-A signaling is critical for establishment and maintenance of the glomerular filtration barrier and strongly supports a pivotal role for VEGF-A in renal disease.
doi:10.1172/JCI200317423
PMCID: PMC151905  PMID: 12618525

Results 1-7 (7)