The recombinant transmembrane protein type XIII collagen is shown to reside on the plasma membrane of insect cells in a 'type II' orientation. Expressions of deletion constructs showed that sequences important for the association of three alpha1(XIII) chains reside in their N- rather than C-terminal portion. In particular, a deletion of residues 63-83 immediately adjacent to the transmembrane domain abolished the formation of disulfide-bonded trimers. The results imply that nucleation of the type XIII collagen triple helix occurs at the N-terminal region and that triple helix formation proceeds from the N- to the C-terminus, in opposite orientation to that of the fibrillar collagens. Interestingly, a sequence homologous to the deleted residues was found at the same plasma membrane-adjacent location in other collagenous transmembrane proteins, suggesting that it may be a conserved association domain. The type XIII collagen was secreted into insect cell medium in low amounts, but this secretion was markedly enhanced when the cytosolic portion was lacking. The cleavage occurred in the non-collagenous NC1 domain after four arginines and was inhibited by a furin protease inhibitor.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80 -85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K m values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä , R. (2000) J. Biol. Chem. 275, 36158 -36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.

Type XIII collagen is a type II transmembrane protein found at many sites of cell adhesion in tissues. Homologous recombination was used to generate a transgenic mouse line (Col13a1 N/N ) that expresses N-terminally altered type XIII collagen molecules lacking the short cytosolic and transmembrane domains but retaining the large collagenous ectodomain. The mutant molecules were correctly transported to focal adhesions in cultured fibroblasts derived from the Col13a1 N/N mice, but the cells showed decreased adhesion when plated on type IV collagen. These mice were viable and fertile, and in immunofluorescence stainings the mutant protein was located in adhesive tissue structures in the same manner as normal ␣1(XIII) chains. In immunoelectron microscopy of wild-type mice type XIII collagen was detected at the plasma membrane of skeletal muscle cells whereas in the mutant mice the protein was located in the adjacent extracellular matrix. Affected skeletal muscles showed abnormal myofibers with a fuzzy plasma membrane-basement membrane interphase along the muscle fiber and at the myotendinous junctions, disorganized myofilaments, and streaming of z-disks. The findings were progressive and the phenotype was aggravated by exercise. Thus type XIII collagen seems to participate in the linkage between muscle fiber and basement membrane, a function impaired by lack of the cytosolic and transmembrane domains. The collagen superfamily of proteins consists of more than 19 types of collagen and several other proteins with collagen-like domains.1 Type XIII collagen and the hemidesmosomal component type XVII collagen form a subfamily of transmembrane collagens. 2 The genes of human and mouse type XIII collagen are 135 to 138 kb in size, consisting of 42 exons, and they are localized to chromosome 10 in both species.3-5 The encoded type XIII collagen consists of three collagenous domains (COL1 to COL3) separated and flanked by four noncollagenous domains (NC1 to NC4). 6,7 The precursor RNAs that encode type XIII collagen undergo complex alternative splicing, which is predicted to affect the structures of the COL1, NC2, and COL3 domains of the human and mouse chains. 5,6,8 -10 Type XIII collagen produced in insect cells forms ␣1(XIII) homotrimers, and the three collagenous domains fold into a stable triple-helical conformation. 11 The type XIII collagen molecules have been shown to reside on the plasma membranes of cells in a type II orientation with a short N-terminal cytosolic portion, a transmembrane domain, and an extensive collagenous ectodomain.12 Sequences that are important for association of the three ␣1(XIII) chains reside in the Nterminal region, and hence triple helix formation is thought to proceed from the N terminus to the C terminus, in the opposite orientation to that known to occur in the fibrillar collagens.12 The extracellular ligands of type XIII collagen have not been identified, but recent studies with recombinant protein demonstrate that its ectodomain interacts strongly with the I domain of ␣1 integrin. ...

Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated a1(XIII) chains. Shortened a1(XIII) chains were synthesized by ®bro-blasts from mutant mice, and the lack of intracellular accumulation in immuno¯uorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myo®laments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development. Keywords: collagen/fetal lethality/placentation/transgenic mice/vascularization IntroductionThe collagen superfamily of proteins consists of >19 types of collagen and several other proteins with collagen-like domains (Vuorio and de Crombrugghe, 1990;Prockop and Kivirikko, 1995). Type XIII collagen and type XVII collagen form a subfamily of membrane-bound collagens (Pihlajaniemi and Rehn, 1995). Type XIII collagen consists of three collagenous domains (COL1±3) separated and¯anked by four non-collagenous domains (NC1±4) (Pihlajaniemi and Tamminen, 1990). It is predicted that the structures of the COL1, NC2 and COL3 domains of the human and mouse type XIII collagen chains are affected by alternative splicing (Pihlajaniemi and Tamminen, 1990;Tikka et al., 1991;Juvonen and Pihlajaniemi, 1992;Juvonen et al., 1992Juvonen et al., , 1993Peltonen et al., 1997).Type XIII collagen produced in insect cells forms a1(XIII) homotrimers, and the three collagenous domains fold into a stable triple helical conformation (Snellman et al., 2000a). The N-terminal NC1 domain contains a highly hydrophobic transmembrane domain, and the type XIII collagen molecules reside on the plasma membrane of cells in a`type II' orientation with a short N-terminal cytosolic portion, a transmembrane domain and an extensive collagenous ectodomain (Snellman et al., 2000b). Sequences that are important for the association of the three a1(XIII) chains reside in the N-terminal region, and triple helix formation is thought to proceed from the N-terminus to the C-terminus, in the opposite orientation to that known to occur in the ®brillar collagens (Snellman et al., 2000b). The ligands of type XIII collagen are not known, but recent studies with recombinant protein demonstrate that it interacts strongly with the I-domain of a1 integrin (Nykvist et al., 2000).Immunohistochemical studies of mature human...