The ancestral gene for transcribed, low-copy repeats in the Prader-Willi/Angelman region encodes a large protein implicated in protein trafficking, which is deficient in mice with neuromuscular and spermiogenic abnormalities

Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.     

Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of alpha3, alpha4, and alpha5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome

Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using pseudolysin (EC 3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 degrees C and the other at 25 degrees C, and their chain composition was determined by use of monoclonal antibodies. The 4 degrees C protomers comprise the alpha1(IV) and alpha2(IV) chains, whereas the 25 degrees C protomers comprised mainly alpha3(IV), alpha4(IV), and alpha5(IV) chains along with some alpha1(IV) and alpha2(IV) chains. The structure of the 25 degrees C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between alpha3(IV), alpha4(IV), and alpha5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the alpha3(IV). alpha4(IV).alpha5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of not only alpha5(IV) chain but also the alpha3(IV) and alpha4(IV) chains in the GBM of patients with Alport syndrome.     

Isolation of cDNA for a novel human protein KNP-I that is homologous to the E. coli SCRP-27A protein from the autoimmune polyglandular disease type I (APECED) region of chromosome 21q22.3

We have isolated cDNA clones for a novel human protein KNP-I from fetal brain and bone marrow cDNA libraries. Northern blot analysis indicated that the KNP-I gene is ubiquitously expressed in various human tissues. Significant homology of the KNP-I protein with Escherichia coli anti-sigma cross-reacting protein (SCRP-27A) (44% identity) and zebrafish (Brachydanio rerio) esl protein (49% identity) suggested that the KNP-I protein may be involved in a basic cellular function. Genomic sequencing revealed that the KNP-I gene consists of seven exons spanning 12 kb. Exon 5 was involved in alternative splicing. The KNP-I gene was mapped between D21S1460 and D21S25 on human chromosome 21q22.3, 26 kb distal to a Not 1 site of D21S1460. Thus, this novel KNP-I gene could be a candidate gene for autoimmune polyglandular disease type I (APECED) and other disorders mapped to this region.