A novel low-light (LL) adapted light-harvesting complex II has been isolated from Rhodopseudomonas palustris. Previous work has identified a LL B800-850 complex with a heterogeneous peptide composition and reduced absorption at 850 nm. The work presented here shows the 850 nm absorption to be contamination from a high-light B800-850 complex and that the true LL light-harvesting complex II is a novel B800 complex composed of eight alpha beta(d) peptide pairs that exhibits unique absorption and circular dichroism near infrared spectra. Biochemical analysis shows there to be four bacteriochlorophyll molecules per alpha beta peptide rather than the usual three. The electron density of the complex at 7.5 A resolution shows it to be an octamer with exact 8-fold rotational symmetry. A number of bacteriochlorophyll geometries have been investigated by simulation of the circular dichroism and absorption spectra and compared, for consistency, with the electron density. Modeling of the spectra suggests that the B850 bacteriochlorophylls may be arranged in a radial direction rather than the usual tangential arrangement found in B800-850 complexes.

Bone morphogenetic protein-1 (BMP-1) is a shorter spliced variant of mammalian tolloid (mTld), both of which cleave the C-propeptides of type I procollagen during the synthesis of extracellular matrix collagen fibrils. The fact that BMP-1 and mTld both exhibit procollagen C-proteinase (PCP) activity and that BMP-1 is the smaller variant might indicate that BMP-1 comprises the minimal required sequences for PCP activity. BMP-1 comprises a metalloproteinase domain, three CUB domains, and an epidermal growth factor (EGF)-like domain, which is located between the second and third CUB (complement components C1r/C1s, the sea urchin protein Uegf, and BMP-1) domains. In this study we showed the following. 1) The CUB1 domain is required for secretion of the molecule. Domain swapping experiments, in which CUB1 and other CUB domains were interchanged, resulted in retention of the proteins by cells. Therefore, CUB1 and its location immediately adjacent to the metalloproteinase domain are essential for secretion of the protein. 2) Mutants lacking the EGFlike and CUB3 domains exhibited full C-proteinase activity. In contrast, mutants lacking the CUB2 domain were poor C-proteinases. 3) Further studies showed that Glu-483 on the ␤4-␤5 loop of CUB2 is essential for Cproteinase activity of BMP-1. In conclusion, the study showed that the minimal domain structure for PCP activity is considerably shorter than expected and comprises the metalloproteinase domain and the CUB1 and CUB2 domains of BMP-1. BMP-11 was isolated in the latter half of the 1980s from osteogenic fragments of bone and was shown to induce cartilage formation in vivo (1, 2). Unlike other BMPs, BMP-1 is a metalloproteinase with a proteinase domain that is homologous to the crayfish enzyme astacin (3) and a C-terminal domain comprising three CUB domains and one EGF-like domain. Subsequent work showed that BMP-1 is a smaller spliced variant of mTld (4). The functions of BMP-1 remained unknown until it was shown that it can cleave chordin, an antagonist of BMP-2, which can direct bone and cartilage formation (5).BMP-1 and mTld exhibit similar substrate specificity in vitro in that they can cleave precursors of extracellular matrix proteins including fibrillar procollagens (6, 7), biglycan (8), type VII procollagen (9), prolysyl oxidase (10, 11), and chains of laminin (12, 13). Evidence from gene knock-out studies in mice in which the mouse tolloid gene was mutated showed that BMP-1 and/or mTld are essential for normal assembly of extracellular matrix (14). Electron microscopy of the skin detected the presence of abnormal collagen fibrils in the knock-out mouse, which showed that BMP-1 and/or mTld are essential for normal cleavage of procollagen.BMP-1 is encoded by mtld, which also gives rise to mammalian tolloid (mTld or vertebrate Tld). mTld contains five CUB domains and two EGF-like domains at its C terminus, whereas BMP-1 lacks the most C-terminal two CUBs and one EGF-like domain. Interestingly, both BMP-1 and mTld can cleave the C-propeptides of fibril-forming proc...

Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomeraselike protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1␣, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn 91 (prodomain), Asn 142 (metalloproteinase domain), Asn 332 and Asn 363 (CUB1 domain), Asn 599 (CUB3 domain), and Asn 726 in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn 726 presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.Bone morphogenetic protein-1 (BMP-1), 1 also known as procollagen C-proteinase-1 (PCP-1) was first identified in osteogenic extracts of bone (1). BMP-1 is a smaller splice variant of mammalian tolloid, which is the vertebrate ortholog of tolloid, in Drosophila. BMP-1, mammalian tolloid, and two highly homologous relatives tolloid-like 1 and 2 constitute the tolloid clade of astacin-like metalloproteinases that have important functions in development and extracellular matrix formation (2). BMP-1 cleaves fibrillar procollagen type I, II, III (3, 4), and V (5-7), as well as type VII procollagen (8), prolysyl oxidase (9), probiglycan (10), and the ␥2 chain of prolaminin 5 (11). BMP-1 also cleaves chordin (2) therefore affecting dorsal-ventral patterning in vertebrates (12). Similarly, tolloid cleaves the chordin homologue short gastrulation during Drosophila embryo development (13). Bmp1 homozygous null mice are perinatal lethal, with defects in ventral body wall closure and collagen fibrillogenesis (14), which illustrates the importance of BMP-1 in tissue assembly and development.BMP-1 consists of a prodomain that is cleaved by a furin-like enzyme in the trans-Golgi network, 2 an astacin-like zinc metalloproteinase domain (15), one epidermal growth factor-like domain, and three CUB domains. In other proteins, CUB domains mediate protein-protein interactions (16).BMP-1 purified from mouse fibroblasts culture medium has been shown to be N-glycosylated (17). Sequence analysis reveals six potential N-glycosylation sites, one of which is located in the prodomain and five in the mature (active) molecule. However, no information is available on the structure and function of the glycosylation sites....