Differential oxidation of HLA-B2704 and HLA-B2705 in lymphoblastoid and transfected adherent cells.

MHC class I molecules are predominantly involved in the presentation of antigens from viral proteins to CD8+ T cells of the immune system. However, MHC proteins can also be linked to autoimmune diseases, and the HLA-B27 allele is expressed by 95% of people with the rheumatic condition ankylosing spondylitis (AS). A precise molecular explanation for the association between HLA-B27 and AS is still lacking, although it is known that inappropriately disulfide bonded HLA-B27 heavy chains can be found at both the cell surface and in the endoplasmic reticulum (ER) of HLA-B27 expressing cells. This papers shows that HLA-B27 heavy chain misfolding does not depend on any unpaired cysteine residue per se when HLA-B27 is highly expressed. Also shown is that major differences exist in the disulfide-dependent conformations of two HLA-B27 subtypes, HLA-B2704 and HLA-B2705. The results imply that residues 77, 152, and/or 211 influence the redox potential of the MHC class I heavy chain and suggest that manipulating the redox environment can alter the conformational state of HLA-B27 subtypes.

PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

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 isomerase-like 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 Ero1alpha, 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-1 (BMP-1). Identification of the minimal domain structure for procollagen C-proteinase activity.

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 EGF-like 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 beta4-beta5 loop of CUB2 is essential for C-proteinase 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.

Post-translational modification of bone morphogenetic protein-1 is required for secretion and stability of the protein.

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.

The 7.5-A electron density and spectroscopic properties of a novel low-light B800 LH2 from Rhodopseudomonas palustris.

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.