Ilse Oberbäumer

The Structure of Type IX Collagen

Investigations of the major structural component of basement membranes have shown for the first time a collagenous protein with a molecular and macromolecular structure different from that of the fiber-forming collagens I, 11, and 111.’ This protein, termed collagen IV, contains certain elements similar in structure to those of the fiber-forming collagens, but not arranged in an analogous fashion. These include a triple helix, nonhelical areas, and globular domains. In order to form a macromolecular network optimally adapted to the highly elastic and mechanically stable sheet-like organization of basement membranes, these elements had to be assembled in a different manner. Collagen IV molecules do not aggregate in a fashion parallel to fibrous structures; they are assembled via their end regions and form a loose network of individual molecules. Basement membranes from bovine kidney glomerulizp3 and bovine lens capsule4,’ or basement membrane rich tissues such as human placentaGg have been used as sources for collagen IV. Of particular advantage was the use of the transplantable EHS mouse which synthesizes basement membrane constituents in large quantities. Successful deduction of the insoluble collagen IV network started with limited proteolytic digestion of such tissues, mainly with pepsin and bacterial collagenase. Electron microscopical characterization of the dissolved fragments and of the intact molecule allowed the reconstruction of the macromolecular structure of the collagen IV n e t ~ o r k . ” ~ ~ ’ ~ Protein chemical investigations of the two a chains of collagen IV were carried out parallel to the electron microscopical characterization and have resulted in the almost complete elucidation of the amino acid sequence of the 1700-residues-long al(1V) chain. In this paper we describe the structural principles of three important domains of the collagen IV molecule: the triple helix that determines the biomechanical properties of the network, and the two terminal regions, the NH,terminal triple-helical 7s domain and the COOH-terminal globular NC1 domain, which are responsible for the assembly of the molecules.

Structure and Biology of the Globular Domain of Basement Membrane Type IV Collagena

A procedure was developed for purifying the globular domain NC1 of basement membrane collagen from collagenase digests of a variety of tissues. The globule (Mr = 170,000) is a hexameric structure originating from two collagen IV molecules that are cross-linked at their COOH-terminal ends. Dissociation into subunits derived from alpha 1(IV) and alpha 2(IV) chains occurs at a pH below 4 and after denaturation (8 M urea). The subunits obtained include monomers (Mr = 28,000) and two different dimers (Da,Db) which are connected by disulfide bonds (Db) and/or nonreducible bonds (Da). Almost perfect reconstitution to hexamers is obtained in neutral buffer with mixtures of the subunits or purified dimers but not with purified monomers. Stabilization by dimer formation and other physical data suggest conformationally distinct segments within the subunits, which is also supported by a repeating subdomain structure deduced from cDNA sequences. Monocline crystals of NC1 give a sufficiently detailed X-ray diffraction pattern that should permit elucidation of the three-dimensional structure of the hexamer. Antibodies raised against the globular domain react with all subunits and mainly recognize epitopes stabilized by internal disulfide bridges and/or the hexameric assembly. Immunoprecipitation tests with these antibodies demonstrated a slightly larger subunit size of NC1 in PYS-2 cell culture and the rapid release of precursor-specific segments prior to secretion from the cells. Autoantibodies against mouse tumor NC1 were produced in mice and were detected both in the blood and as tissue-bound forms (kidney, lung). The autoantibody response is accompanied by certain pathological alterations mimicking Goodpastures syndrome. The possible relationship between the two diseases is substantiated by reaction of Goodpasture antisera with the globular domain obtained from various tissue sources.

New pUC-derived expression vectors for rapid construction of cDNA libraries

We have constructed a new series of the pUC-derived plasmids with an extended polylinker obtained from M13tg131 [Kieny et al., Gene 26 (1983) 91-99]. These vectors allowed us to design a simplified version of the method of Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] for establishing complete cDNA libraries. Improvements included easy recovery of the inserted cDNA due to the extended polylinkers; use of these vectors for gene expression in Escherichia coli, and amenability to supercoil sequencing with the universal primers of the M13 system [Chen and Seeburg, DNA 4 (1985) 165-170], which speeds up the identification of positive clones. Moreover, there is no need for an additional linker fragment, as was required by the Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] method. We have successfully used this system to obtain cDNA clones coding for the different chains of the large basement membrane proteins type IV collagen and laminin.

cDNA and protein sequence of the NC1 domain of the α2-chain of collagen IV and its comparison with α1(IV)

We present the complete cDNA and derived amino acid sequence of the non‐collagenous domain NCl of α2(IV). Comparison with the corresponding NC1 domain of α(IV) reveals a high degree of homology at the protein level in contrast to the barely homologous triple‐helical sequences of both chains.

DNA fingerprinting with oligonucleotides can differentiate cell lines derived from the same tumor

SummaryOligonucleotide fingerprinting was applied to investigate the relatedness of several cell lines that were established between 1973 and 1977 from a teratocarcinoma. We were able to distinguish cell lines derived at different times. In addition, sublines from one cell line (PYS-2) could be discriminated by using a combination of different probes. Therefore multilocus fingerprinting with oligonucleotides is a useful method for monitoring changes in cell lines kept in culture for many generations.

Evaluation of mRNA steady-state and protein levels for basement membrane proteins in cultured murine cells

SummaryWe have determined the mRNA steady-state levels for the six constituent polypeptide chains of the basement membrane proteins collagen IV, laminin and nidogen in murine cell lines derived from a teratocarcinoma, and in some other cell lines of different origin in stationary cultures and during different growth phases. The mRNA and protein levels change in response to growth phase. The amounts of the mRNAs for the single chains do not agree with the ratios needed for the different peptide chains of collagen IV and laminin. While the mRNA and protein levels for laminin are in a similar range for the teratocarcinoma-derived cell lines, the mRNA and protein levels vary by at least a factor of 10 for collagen IV. These results point to complex posttranscriptional regulatory mechanisms for the biosynthesis of basement membrane proteins.

Detection of RNA on Northern blots by negative staining with aurintricarboxylic acid

Aurintricarboxylic acid (ATA) is a well-known inhibitor of RNA and DNA modifying enzymes and was suggested as a potent RNase inhibitor for preparation of RNA (Hallick et al., 1977, Nucleic Acids Res. 4, 3055-3064). We show that ATA is a very useful stain for detecting RNA on Northern blots and slot blots although it did not fully protect purified RNA in concentrated solution against RNase A.

Chromatographic isolation and characterization of streptogramin antibiotics

Abstract High-performance and medium-pressure liquid chromatographic techniques were developed for the rapid isolaiton, identification and characterization of the components from crude virginiamycin and viridogrisein. Two new minor components, denoted virginiamycin S5 and viridogrisein II, were isolated. Their structures were determined by amino acids analysis and mass spectrometry. (I. Oberbaumer et al., Helv. Chim. Acta, 65 (1982) 2280).