Paul Cullen

Production of Type VI Collagen by Human Macrophages: A New Dimension in Macrophage Functional Heterogeneity

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.

IsoSVM – Distinguishing isoforms and paralogs on the protein level

BackgroundRecent progress in cDNA and EST sequencing is yielding a deluge of sequence data. Like database search results and proteome databases, this data gives rise to inferred protein sequences without ready access to the underlying genomic data. Analysis of this information (e.g. for EST clustering or phylogenetic reconstruction from proteome data) is hampered because it is not known if two protein sequences are isoforms (splice variants) or not (i.e. paralogs/orthologs). However, even without knowing the intron/exon structure, visual analysis of the pattern of similarity across the alignment of the two protein sequences is usually helpful since paralogs and orthologs feature substitutions with respect to each other, as opposed to isoforms, which do not.ResultsThe IsoSVM tool introduces an automated approach to identifying isoforms on the protein level using a support vector machine (SVM) classifier. Based on three specific features used as input of the SVM classifier, it is possible to automatically identify isoforms with little effort and with an accuracy of more than 97%. We show that the SVM is superior to a radial basis function network and to a linear classifier. As an example application we use IsoSVM to estimate that a set of Xenopus laevis EST clusters consists of approximately 81% cases where sequences are each others paralogs and 19% cases where sequences are each others isoforms. The number of isoforms and paralogs in this allotetraploid species is of interest in the study of evolution.ConclusionWe developed an SVM classifier that can be used to distinguish isoforms from paralogs with high accuracy and without access to the genomic data. It can be used to analyze, for example, EST data and database search results. Our software is freely available on the Web, under the name IsoSVM.

Evidence for an alternative genomic structure, mRNA and protein sequence of human ABCA13.

ABC transporters form one of the major families of transport proteins. In humans, the ABC family comprises seven subfamilies named A to G, of which the A subfamily contains twelve members. Among these are several well-characterized transporters, including ABCA1, which is involved in cellular cholesterol transport and HDL formation, and ABCA4, which is a transporter for vitamin A derivatives in photoreceptor cells. The function of another subfamily member termed ABCA13 is unknown. The human ABCA13 gene has been reported to span 450kb of genomic DNA at chromosomal locus 7p12.3 and to encode a 5058 amino acid protein that includes two unusually large exons close to the N-terminus. We now show that the gene as well as the corresponding mRNA and protein may be considerably shorter than previously thought. We used PCR and RACE to identify a genomic sequence spanning about 350kb and encoding a protein of 2323 amino acids. This corresponds to the C-terminal half of the previously reported ABCA13 protein but lacks the residues reportedly encoded by the two very big N-terminal exons. Using immunoprecipitation and Western blot analyses we identified a protein of about 260kDa in size likely representing the shorter protein proposed here. Computer analyses showed that our proposed sequence contains all the structural elements of an ABCA protein and agrees well with the mouse ABCA13 protein sequence. Additionally, we identified a putative promoter region containing well-conserved TATA and CAAT boxes just upstream of our transcription start site. Overall, our data provide good evidence for an alternative human ABCA13 transcript and protein.