Mon-Li Chu

Recently cDNA clones encoding the entire humanalpha1XVI chain have been isolated and characterized. The overallstructural arrangement of the deduced polypeptide differs significantlyfrom other known collagen types. However, this polypeptide exhibitscertain structural features characteristically seen in members of thefibril associated collagen, types IX, XII and XIV. It also resembles thecuticle collagen of C elegans in that both collagens contain similarcysteine-rich motifs. In order to elucidate the biological function ofthis collagen, we propose the following specific aims. 1 Preparationand characterization of antibodies specific for the human alpha1XVIcollagen using synthetic peptides derived from the cDNA sequence andrecombinant proteins produced by expressing cDNA in cultured cells. 2Characterization of type XVI collagen in cultured cells and tissues. Themolecular size and chain composition of the native type XVI collagen willbe determined by immunoblotting and immunoprecipitation using antibodiesprepared above. 3 Elucidation of the spatial and temporal expressionof the alpha1XVI collagen. The mRNA and protein expression in cellsand tissues from humans and mice will be examined by RNA hybridizationand immunofluorescence staining. 4 Elucidation of thestructurefunction relationship of the alpha1VI collagen using proteinfragments, synthetic peptides and recombinant proteins produced in themammalian expression system. 5 Characterization of other constituentchains of type XVI collagen if studies proposedabove indicate that itis a heterotrimer. 6 Isolation and characterization of the mousealpha1XVI collagen gene and use the genomic fragment to developtransgenic mice with homozygous disruption of the alpha1XVI collagengene.Tssue Culture Core facilities are of central importance for thisProgram Project, as they will continue to provide cultured cells fromindividuals affected with EB, from their unaffected family members, andfrom unrelated healthy controls for study in all Projects. During theprevious years of support, the tissue culture facility has handled alarge number of human cells from EB and control subjects. It has alsomaintained a large number of either spontaneously or virally transormedhuman cell lines that have been extremely helpful in elucidating geneexpression of BMZ macromolecules, including type VII collagen. Theseestablished cell lines include a human amniotic endothelial cell lineWISH, a human epidermoid oral carcinoma cell line KB, and a humanfibrosarcomacell line HT-1080, which are commercially availableATCC.The goal of this project is to elucidate protein and gene structures fornovel components of the cutaneous basement membrane zone. Novel proteinsof the skin basement membrane zone are constantly being discovered. Theinformation on these new proteins and genes is fundamental to the studyof mutations in EB During the past four years, we have characterizeda number of normal basement membrane genes. The gene probes developedin this project are instrumental in the molecular characterization of EBmutations. In this renewal application, we propose to use the samestrategies to characterize additional basement membrane zone genes. Ourinitial efforts will be directed toward completing the current studieson the genes for the 230-kDa and 180-kDa bullous pemphigoid antigensBPAG1 and BPAG2, respectively. At the same time, we will continue thework we have initiated towards characterizing a new basemetn membraneprotein, BM90, and a related gene that we have discovered during thecourse of this study. We will then proceed to isolate cDNA and genomicclones for one or more novel components in skin such as type XIVcollagen, kalininepiligrin, or other new proteins in the skin basementmembrane zone.VI collagen is a microfibrillar component found in most soft tissuesand in cartilage. The triple-helical molecules are composed of threeconstituent chains, alpha2VI, alpha2VI and alpha3VI chains. Several polymorphic mRNAs encoding for alpha2VI and alpha3VI variantsare transcribed from the genes by alternative splicing mechanism. Thelong term objectives of this grant are to elucidate the biologicalfunction of collagen VI and to investigate whether defects in collagen VIare associated with heritable or acquired connective tissue diseases. The specific aims of this competing continuation application are: 1 Todefine the biological significance of the variation in mRNAs for thealpha2VI and alpha3VI chains. The distribution of mRNA and proteinvariants will be determined in human cell lines and mouse tissues ofdifferent developmental stages. 2 To define the molecular requirementsfor the assembly of the collagen VI triplehelical molecules. Theapproach will be to express full-length or partial cDNAs of each chaininmammalian cells and to study the assembly of the triple-helices. 3 Todefine the functional role of specific domains in each of the threecollagen VI chains. Fusion proteins containing specific domains andvariant domains will be prepared in bacterial or mammalian cellsexpression vectors and used for functional studies. 4 To examine thecomplexity of collagen VI structure in tissues by searching for isoformsof collagen VI chains and for associated noncollagenous proteins. 5 Tocomplete the isolation of genomic clones for the alphalVI, alpha2VIand alpha3VI collagen genes and to define the important structuralfeatures of the genes. 6 To identify cis- and trans-regulatory elementsthat control the expression of the three collagen VI genes.