Collagen IV of basement membranes: I. Origin and diversification of COL4 genes enabling animal evolution.

Collagen IV is a primordial component of basement membranes, a specialized form of extracellular matrix that enabled multi-cellular epithelial tissues. In mammals, collagen IV assembles from a family of six α-chains (α1 to α6), encoded by six genes (COL4A1 to COL4A6), into three distinct scaffolds: the α121, the α345 and a mixed scaffold containing both α121 and α565. The six mammalian COL4A genes occur in pairs that occur in a head-to-head arrangement on three distinct chromosomes. In Alport syndrome, variants in the COL4A3, 4 or 5 genes cause either loss or defective assembly of the collagen IV α345 scaffold which results in a dysfunctional glomerular basement membrane, proteinuria and progression to renal failure in millions of people worldwide. Here, we determine the evolutionary emergence and diversification of the COL4A genes using comparative genomics and biochemical analyses. Using syntenic relationships to genes closely linked to the COL4A genes, we determine that the COL4A3 and COL4A4 gene pair appeared in cyclostomes (hagfish and lampreys) while the COL4A5 and COL4A6 gene pair emerged in gnathostomes, jawed vertebrates. The more basal chordate species, lancelets and tunicates, do not have discrete kidneys and have a single COL4A gene pair, though often with single isolated COL4 genes similar to those found in C elegans . Remarkably, while the six COL4A genes are conserved in vertebrates, amphibians have lost the COL4A3 and COL4A4 genes. Our findings of the evolutionary emergence of these genes, together with the amphibian double-knockout, opens an experimental window to gain insights into functionality of the Col IV α345 scaffold.

Unicellular ancestry and mechanisms of diversification of Goodpasture antigen-binding protein.

The emergence of the basement membrane (BM), a specialized form of extracellular matrix, was essential in the unicellular transition to multicellularity. However, the mechanism is unknown. Goodpasture antigen-binding protein (GPBP), a BM protein, was uniquely poised to play diverse roles in this transition owing to its multiple isoforms (GPBP-1, -2, and -3) with varied intracellular and extracellular functions (ceramide trafficker and protein kinase). We sought to determine the evolutionary origin of GPBP isoforms. Our findings reveal the presence of GPBP in unicellular protists, with GPBP-2 as the most ancient isoform. In vertebrates, GPBP-1 assumed extracellular function that is further enhanced by membrane-bound GPBP-3 in mammalians, whereas GPBP-2 retained intracellular function. Moreover, GPBP-2 possesses a dual intracellular/extracellular function in cnidarians, an early nonbilaterian group. We conclude that GPBP functioning both inside and outside the cell was of fundamental importance for the evolutionary transition to animal multicellularity and tissue evolution.

A comparative analysis of microbial profile of Guinea fowl and chicken using metagenomic approach.

Probiotics are live microbial feed supplements that promote growth and health to the host by minimizing non-essential and pathogenic microorganisms in the host's gastrointestinal tract (GIT). The campaign to minimize excessive use of antibiotics in poultry production has necessitated development of probiotics with broad application in multiple poultry species. Design of such probiotics requires understanding of the diversity or similarity in microbial profiles among avian species of economic importance. Therefore, the objective of this research was to establish and compare the microbial profiles of the GIT of Guinea fowl and chicken and to establish the microbial diversity or similarity between the two avian species. A metagenomic approach consisting of the amplification and sequence analysis of the hypervariable regions V1-V9 of the 16S rRNA gene was used to identify the GIT microbes. Collectively, we detected more than 150 microbial families. The total number of microbial species detected in the chicken GIT was higher than that found in the Guinea Fowl GIT. Our studies also revealed phylogenetic diversity among the microbial species found in chicken and guinea fowl. The phylum Firmicutes was most abundant in both avian species whereas Phylum Actinobacteria was most abundant in chickens than Guinea fowls. The diversity of the microbial profiles found in broiler chickens and Guinea fowls suggest that the design of effective avian probiotics would require species specificity.

Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues.

The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as one of the earliest-branching extant animal phyla, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here, we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of non-bilaterian animal phyla. We identified basement membrane (BM) and collagen IV in Ctenophora, and show that the structural and genomic features of collagen IV are homologous to those of non-bilaterian animal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Moreover, collagen IV is absent in unicellular sister-groups. Collectively, we conclude that collagen IV and its variant, spongin, are primordial components of the extracellular microenvironment, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis.

Pyridoxamine protects proteins from damage by hypohalous acids in vitro and in vivo.

Diabetes is characterized, in part, by activation of toxic oxidative and glycoxidative pathways that are triggered by persistent hyperglycemia and contribute to diabetic complications. Inhibition of these pathways may benefit diabetic patients by delaying the onset of complications. One such inhibitor, pyridoxamine (PM), had shown promise in clinical trials. However, the mechanism of PM action in vivo is not well understood. We have previously reported that hypohalous acids can cause disruption of the structure and function of renal collagen IV in experimental diabetes (K.L. Brown et al., Diabetes 64:2242-2253, 2015). In the present study, we demonstrate that PM can protect protein functionality from hypochlorous and hypobromous acid-derived damage via a rapid direct reaction with and detoxification of these hypohalous acids. We further demonstrate that PM treatment can ameliorate specific hypohalous acid-derived structural and functional damage to the renal collagen IV network in a diabetic animal model. These findings suggest a new mechanism of PM action in diabetes, namely sequestration of hypohalous acids, which may contribute to known therapeutic effects of PM in human diabetic nephropathy.

Molecular tools to support metabolic and immune function research in the Guinea Fowl (Numida meleagris).

BACKGROUND: Guinea fowl (Numidia meleagris) production as an alternative source of meat and poultry has shown potential for economic viability. However, there has been little progress in characterizing the transcriptome of the guinea fowl. In this study RNA-sequencing and de novo transcriptome assembly of several Guinea fowl tissues (pancreas, hypothalamus, liver, bone marrow and bursa) which play key roles in regulating feed intake, satiety, and immune function was performed using Illumina's Hi-Seq 2000. RESULTS: 74 million sequences were generated and assembled into 96,492 contigs using the Trinity software suite. Over 39,000 of these transcripts were found to have in silico translated protein sequences that are homologous to chicken protein sequences. Gene ontology analysis uncovered 416 transcripts with metabolic functions and 703 with immune function. CONCLUSION: The transcriptome information presented here will support the development of molecular approaches to improve production efficiency of the guinea fowl and other avian species.

Hypohalous acids contribute to renal extracellular matrix damage in experimental diabetes.

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid-derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1ß1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W(28) and W(192) are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid-derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.

Expression of Potential Regulatory Genes in Abdominal Adipose Tissue of Broiler Chickens during Early Development.

The identities of genes that underlie population variation in adipose tissue development in farm animals are poorly understood. Previous studies in our laboratory have suggested that increased fat tissue involves the expression modulation of an array of genes in broiler chickens. Of special interest are eight genes, FGFR3, EPHB2, IGFBP2, GREM1, TNC, COL3A1, ACBD7, and SCD. To understand their expression regulation and response to dietary manipulation, we investigated their mRNA levels after dietary manipulation during early development. Chickens were fed either a recommended standard or a high caloric diet from hatch to eight weeks of age (WOA). The high caloric diet markedly affected bodyweight of the broiler birds. mRNA levels of the eight genes in the abdominal adipose tissue were assayed at 2, 4, 6, and 8 WOA using RT-qPCR. Results indicate that (1) FGFR3 mRNA level was affected significantly by diet, age, and diet:age interaction; (2) COL3A mRNA level was repressed by high caloric diet; (3) mRNA levels of EPHB2, ACBD7, and SCD were affected by age; (4) mRNA level of TNC was modulated by age:diet interaction; (5) changes in GREM1 and IGFBP2 mRNA levels were not statistically different.