Tilman Voss

Macromolecular organization of natural and recombinant lung surfactant protein SP 28–36: Structural homology with the complement factor C1q

The macromolecular structure of the pulmonary surfactant apolipoprotein SP 28-36 has been determined. For SP 28-36 isolated from dog lung lavage, a flower bouquet-like hexameric structure with six globular domains connected by short stalks to a common stem was revealed by electron microscopy, using the rotary shadowing technique. This structure is very similar to that published for the subcomponent C1q of the first component of complement C1. The lavage material was compared with the homologous human recombinant SP 28-36 by the same technique. Mostly smaller aggregates like di-, tri- and tetramers as well as very high aggregates were observed. Mild reduction of the recombinant material revealed the lollipop-shaped monomers composed of a globular domain and a tail with a discrete kink in the middle portion. The collagenous nature of the tail was demonstrated by circular dichroism spectroscopy. This implies that the mammalian expression system assembles the monomeric subunits correctly. Assembly into the hexameric structures, however, does not proceed quantitatively.

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.

Lung surfactant: A biotechnological challenge

The genes for all three of the bona fide surfactant associated proteins have been cloned, allowing their production by recombinant DNA technology. In addition, improved protocols for the isolation of the natural surfactant proteins (NSP) made them available in larger quantities. Whereas, the NSP are often mixtures of allelic variants or functional isomers from gene families, the recombinant proteins (RSP) are obtained as single pure protein species. Antibodies directed against the N/RSP in combination with DNA probes have allowed new approaches to analyze the formation, location, transport, structure and functional capacities of these molecules as well as their interactions with one another and the phospholipids.