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Differential roles for laminins in intestinal homeostasis
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
204 Structural studies of breaks in the type IV collagen triple helix A. Mohs a, C. Strafaci a , Y. Li b, J. Baum b, B. Brodsky a b
b
UMDNJ-Robert Wood Johnson Medical School, NJ Rutgers University, NJ
Although the (Gly-X-Y)n repeating sequence is a requirement to form a collagen triple-helix, this pattern is perfectly maintained in some collagen domains but not in others. Type IV collagen contains more than 20 breaks in the (Gly-X-Y)n pattern of its ∼1350 residue long triple-helix. Studies are in progress to systematically classify and analyze breaks within chains of type IV collagen. Statistical analysis shows that residues within the shorter breaks differ from the normal composition of the X and Y residues in the Gly-X-Y repeat, and sequence alignment of the heterotrimeric type IV molecules indicate that most of the interruptions are aligned, often with specific types of paired interruptions. Biophysical studies have been carried out on collagen peptides which model two different types of α5(IV) breaks. Peptides containing a GAAVMG interruption (classified as G4G) and a GFG break (classified as G1G) were found to form stable homotrimeric triple-helices. Differential scanning calorimetry and circular dichroism spectroscopy indicate a dramatic drop in the calorimetric enthalpy and a significant drop in thermal stability as a result of the interruption. NMR studies on specific 15N-labeled residues indicate a normal triple helical structure flanking the breaks, and the presence of ordered structure with altered hydrogen bonding and exchange rates within the breaks. Further studies are designed to clarify the importance of consensus residues within the breaks on structural features and the complementary nature of heterotrimeric break sites. doi:10.1016/j.matbio.2006.08.227
205 Determining the mechanism of integrin-laminin binding M.P. Harris, H. Yamashita, M. Tripathi, V. Quaranta Cancer Biology Department, Vanderbilt University, Nashville, TN 37235 Laminin-5 (Ln-5) is a main component of epithelial basement membranes, where it serves a dual function: Ln-5 can promote either static cell adhesion or cell migration depending on its post-translation modifications. Much of this regulation appears to reside in its cellular receptors. Ln-5 interacts with at least two members of the integrin family of adhesion receptors: integrin α3β1 (stimulates migratory cell
S83
machinery), and α6β4 (supports static adhesion and hemidesmosomes). Ln-5 interacts with these integrins through one or more of its five laminin globular (LG) domains. LG3 is known to interact with the integrins, although the mechanism by which they bind is currently unknown. Data from our lab indicates LG4 can also promote cell adhesion and migration in an integrin dependant manner. We have purified LG3 and LG4 in both mammalian and E. coli expression systems, and a structure/function analysis of these domains is currently underway. We will present structural data that begin to define the molecular details of binding interactions between the integrins and LG3/LG4. doi:10.1016/j.matbio.2006.08.228
206 Differential roles for laminins in intestinal homeostasis Z.X. Mahoney, T.S. Stappenbeck, J.H. Miner Washington Univesity School of Medicine, St. Louis, MO, United States Laminin describes a family of heterotrimeric glycoproteins that are integral components of the basement membrane (BM). The intestinal BM is rich in laminin-511 (α5β1γ1) and underlies intestinal epithelial cells (IECs), which are organized into repeated crypt-villus units. IECs are born in crypts and then migrate onto villi while completing differentiation. IECs apoptose at the tops of villi and are shed into the lumen. Here, we introduced a widely expressed laminin α5 transgene (Mr5) onto a laminin α5 knockout background (Lama5−/−) to rescue developmental defects of Lama5−/− mice. However, Mr5 expression is very low in the adult small intestine, which leads to a compensatory increase in laminin α1 in the villus BM of Lama5−/−; Mr5 mice. This model allows us to study the differential roles of laminin α1 and ?α5 in small intestinal morphogenesis and homeostasis in vivo.In Lama5−/−; Mr5 mice, villi appear to coalesce in the distal small intestine to form cerebroid (like the surface of the brain) or mosaic (individual villi no longer present) patterns. In addition, there are alterations in capillary patterning in the lamina propria. These morphologic patterns strongly resemble that of colon, where laminin α1, rather than α5, is the predominant α chain. The substitution of laminin α1 for α5 affects all aspects of intestinal homeostasis: the proliferative zone is elongated, cell migration rate is increased, apoptosis is reduced, and cell differentiation is perturbed. Altogether, these results suggest that laminin α5 and α1 play crucial and differential roles in organizing crypt-villus architecture, and in regulating diverse cellular events in vivo. doi:10.1016/j.matbio.2006.08.229
204 Structural studies of breaks in the type IV collagen triple helix A. Mohs a, C. Strafaci a , Y. Li b, J. Baum b, B. Brodsky a b
b
UMDNJ-Robert Wood Johnson Medical School, NJ Rutgers University, NJ
Although the (Gly-X-Y)n repeating sequence is a requirement to form a collagen triple-helix, this pattern is perfectly maintained in some collagen domains but not in others. Type IV collagen contains more than 20 breaks in the (Gly-X-Y)n pattern of its ∼1350 residue long triple-helix. Studies are in progress to systematically classify and analyze breaks within chains of type IV collagen. Statistical analysis shows that residues within the shorter breaks differ from the normal composition of the X and Y residues in the Gly-X-Y repeat, and sequence alignment of the heterotrimeric type IV molecules indicate that most of the interruptions are aligned, often with specific types of paired interruptions. Biophysical studies have been carried out on collagen peptides which model two different types of α5(IV) breaks. Peptides containing a GAAVMG interruption (classified as G4G) and a GFG break (classified as G1G) were found to form stable homotrimeric triple-helices. Differential scanning calorimetry and circular dichroism spectroscopy indicate a dramatic drop in the calorimetric enthalpy and a significant drop in thermal stability as a result of the interruption. NMR studies on specific 15N-labeled residues indicate a normal triple helical structure flanking the breaks, and the presence of ordered structure with altered hydrogen bonding and exchange rates within the breaks. Further studies are designed to clarify the importance of consensus residues within the breaks on structural features and the complementary nature of heterotrimeric break sites. doi:10.1016/j.matbio.2006.08.227
205 Determining the mechanism of integrin-laminin binding M.P. Harris, H. Yamashita, M. Tripathi, V. Quaranta Cancer Biology Department, Vanderbilt University, Nashville, TN 37235 Laminin-5 (Ln-5) is a main component of epithelial basement membranes, where it serves a dual function: Ln-5 can promote either static cell adhesion or cell migration depending on its post-translation modifications. Much of this regulation appears to reside in its cellular receptors. Ln-5 interacts with at least two members of the integrin family of adhesion receptors: integrin α3β1 (stimulates migratory cell
S83
machinery), and α6β4 (supports static adhesion and hemidesmosomes). Ln-5 interacts with these integrins through one or more of its five laminin globular (LG) domains. LG3 is known to interact with the integrins, although the mechanism by which they bind is currently unknown. Data from our lab indicates LG4 can also promote cell adhesion and migration in an integrin dependant manner. We have purified LG3 and LG4 in both mammalian and E. coli expression systems, and a structure/function analysis of these domains is currently underway. We will present structural data that begin to define the molecular details of binding interactions between the integrins and LG3/LG4. doi:10.1016/j.matbio.2006.08.228
206 Differential roles for laminins in intestinal homeostasis Z.X. Mahoney, T.S. Stappenbeck, J.H. Miner Washington Univesity School of Medicine, St. Louis, MO, United States Laminin describes a family of heterotrimeric glycoproteins that are integral components of the basement membrane (BM). The intestinal BM is rich in laminin-511 (α5β1γ1) and underlies intestinal epithelial cells (IECs), which are organized into repeated crypt-villus units. IECs are born in crypts and then migrate onto villi while completing differentiation. IECs apoptose at the tops of villi and are shed into the lumen. Here, we introduced a widely expressed laminin α5 transgene (Mr5) onto a laminin α5 knockout background (Lama5−/−) to rescue developmental defects of Lama5−/− mice. However, Mr5 expression is very low in the adult small intestine, which leads to a compensatory increase in laminin α1 in the villus BM of Lama5−/−; Mr5 mice. This model allows us to study the differential roles of laminin α1 and ?α5 in small intestinal morphogenesis and homeostasis in vivo.In Lama5−/−; Mr5 mice, villi appear to coalesce in the distal small intestine to form cerebroid (like the surface of the brain) or mosaic (individual villi no longer present) patterns. In addition, there are alterations in capillary patterning in the lamina propria. These morphologic patterns strongly resemble that of colon, where laminin α1, rather than α5, is the predominant α chain. The substitution of laminin α1 for α5 affects all aspects of intestinal homeostasis: the proliferative zone is elongated, cell migration rate is increased, apoptosis is reduced, and cell differentiation is perturbed. Altogether, these results suggest that laminin α5 and α1 play crucial and differential roles in organizing crypt-villus architecture, and in regulating diverse cellular events in vivo. doi:10.1016/j.matbio.2006.08.229