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M Pfäffle

Publications and source records attributed to M Pfäffle.

11 recordsLinked to original sources
PubMed1992

Selective binding of anchorin CII (annexin V) to type II and X collagen and to chondrocalcin (C-propeptide of type II collagen). Implications for anchoring function between matrix vesicles and matrix proteins.

Anchorin CII is a collagen binding protein of the annexin family associated with plasma membranes of chondrocytes, osteoblasts, and many other cells. As a major constituent of cartilage-derived matrix vesicles it has been shown to bind to native type II and X collagen. In accordance with this observation, here we show the localization of anchorin CII in the extracellular matrix of calcifying cartilage in the fetal human growth plate, and that it was restricted to the chondrocyte surface in proliferating and resting cartilage. Furthermore, we present evidence, using a slot blot assay, that anchorin CII not only binds to native type II and X collagen, but also to chondrocalcin, the carboxy-terminal extension of type II procollagen, in a calcium-independent manner. Pepsin digestion of type II collagen results in loss of anchorin CII binding, confirming our previous notion that the telopeptide region of type II collagen carries anchorin CII binding sites.

Animals↗
PubMed1990

Anchorin CII, a collagen-binding chondrocyte surface protein of the calpactin family.

In an attempt to identify collagen-binding proteins on the chondrocyte surface, a protein of Mr 34KD, called Anchorin CII was isolated from chondrocyte membranes by affinity chromatography on type II collagen sepharose (Mollenhauer & von der Mark, 1983). The protein was localized on the chondrocyte surface by immunofluorescence labeling using a specific rabbit antibody (Mollenhauer et al., 1984), by immunogold labeling and by cell surface iodination (Pfäffle et al., 1988). Fab'fragments of anti anchorin CII reduced the binding of chondrocytes to type II collagen substrates (Mollenhauer et al., 1984). Analysis of the complete primary structure of anchorin CII revealed 4 repetitive domains of each 70-80 amino acid residues, and the absence of hydrophobic transmembrane sequences or signal peptides (Fernández et al., 1988). Thus, anchorin CII is another member of the calpactin/lipocortin/annexin family, although most other members of this family are located strictly intracellularly. Similar to lipocortin I, however, anchorin CII can be identified extracellularly, e.g. in the culture medium of chondrocytes and fibroblasts (Pfäffle et al., 1988). Here we report on further studies on sequence homologies to other annexins, and on the Ca(++)- and phospholipide binding of this protein.

Amino Acid Sequence↗
PubMed1988

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane.

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.

Amino Acid Sequence↗
PubMed1988

Biosynthesis, secretion and extracellular localization of anchorin CII, a collagen-binding protein of the calpactin family.

The amino acid sequence of anchorin CII, a collagen-binding protein isolated originally from chondrocyte membranes, was previously determined by sequencing of cDNA and proteolytic fragments of the protein. Computer analysis of the protein sequence revealed four internal repeats of approximately 70-80 residues, each containing a highly conserved consensus sequence of 17 residues. These repeats show considerable homology with sequences in human and bovine calpactin, lipocortin, endonexin and protein II, which are members of a family of Ca2+- and phospholipid-binding proteins, as well as major substrates of tyrosine kinases. While these proteins have been located at the inner side of the plasma membrane of fibroblasts and epithelial cells, here we present experimental evidence that anchorin CII is at least partially released from cells and binds to the outer cell surface. Biosynthesis studies in cell-free systems and in cell culture indicate that anchorin CII is not processed, which is consistent with the absence of signal sequences from the protein. Yet, pulse-chase experiments show that anchorin is released into the culture medium of fibroblasts after 30 min, and in chondrocyte cultures after 20 h. Anchorin CII was located to the outer cell surface of chondrocytes by lactoperoxidase-catalyzed cell surface iodination as well as by antibody labeling both at light- and electron-microscopical level. The pericellular localization of anchorin CII is consistent with the notion that this protein is involved in the interaction of chondrocytes and fibroblasts with extracellular collagen.

Amino Acid Sequence↗
PubMed1988

Retention of carboxypropeptides in type-II collagen fibrils in chick embryo chondrocyte cultures.

An antibody reacting with the C-propeptide of chick type-II procollagen was used in an attempt to localize this terminal extension of the procollagen molecule (by immunogold labelling) during early collagen fibrillogenesis in chondrocyte cultures. After 2 days in culture the chondrocytes were surrounded by pericellular type-II collagen, as demonstrated by an indirect immunofluorescence labelling technique. An electron microscopy study of these cultures showed that the collagen fibrils were thin (approximately 15 nm diameter), with a poorly visible cross striation, sometimes enhanced by slight thickenings. The antibody against the C-propeptide of type-II procollagen labelled most of the collagen fibrils, according to a very regular pattern constituting a 60 nm periodicity. After 3 days the label was still present on the pericellular collagen fibrils but disappeared from the collagen fibrils of the extracellular matrix. Our results indicate that the C-propeptide of type-II procollagen is retained in the newly formed fibrils.

Animals↗
PubMed1988

Collagen-binding proteins of rat mammary tumor epithelial cells: a biochemical and immunological study.

Collagen-binding proteins were studied in mammary epithelial cells of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. These proteins can be solubilized from cell membranes with 0.1% Triton. Using affinity chromatography on type I collagen-Sepharose and polyacrylamide slab gel electrophoresis, three major proteins of 34,000, 36,000, and 38,000 Da were found. Similar proteins were also present in several other cell types, including both epithelial and mesenchymal cells. Pulse-chase experiments did not indicate a precursor-product relationship of these proteins. Tryptic/chymotryptic peptide maps, however, revealed that the 36,000- and 38,000-Da proteins are very similar but are quite different from the 34,000-Da molecular form. The distribution and function of these proteins were then analyzed by using polyclonal antibodies directed against the entire set of major proteins. In immunofluorescence studies we observed a dense, punctate distribution of fluorescence on the cell surface of isolated and unfixed epithelial organoids and a bright pericellular staining in cultures after fixation. Treatment with the antiserum did not affect attachment and spreading of cuboidal mammary cells to plastic or to a collagen substratum. However, when the antiserum was added to the medium of growing cuboidal cells, it caused the formation of duct-like structures. These studies indicate that collagen-binding proteins may play a role in mammary gland morphology.

Animals↗
PubMed1988

A defective cell surface collagen-binding protein in dermatosparactic sheep fibroblasts.

Fibroblasts from dermatosparactic sheep fail to contract collagen gels and show a reduced attachment to collagenous substrates. By comparing collagen-binding membrane proteins of normal (+/+), homozygote (-/-), and heterozygote (+/-) fibroblasts, we present evidence that the interaction of normal fibroblasts with native type I collagen involves a protein of apparent Mr = 34,000 which is absent from dermatosparactic fibroblasts and seems to be related to anchorin CII. This conclusion was reached from the following experiments: (a) On a blot of membrane proteins from normal fibroblasts radioactively labeled type I collagen bound predominantly to a protein band of 34 kD; dermatosparactic membranes revealed only a small amount of binding to a component with a molecular mass of 47 kD. (b) After separation of normal fibroblast membrane proteins on type I collagen-Sepharose, a collagen-binding component of 34 kD was found which was absent from the corresponding fraction of dermatosparactic membranes. (c) Antibodies to anchorin CII stained the surface of normal (+/+), but not of dermatosparactic (-/-) fibroblasts and labeled a 34-kD component after immunoblotting of normal fibroblast membrane proteins. (d) After metabolic labeling of fibroblasts with [35S]methionine and immunoprecipitation with anti-anchorin CII, 40- and 34-kD components were precipitated from extracts of normal fibroblasts, while the latter component was absent from affected cells. Similar differences were found after immunoblotting of membranes from whole normal or affected skin. These data indicate that dermatosparaxis of sheep involves a molecular defect of a collagen-binding protein. Therefore this disease represents a model to study the complex interaction of cells with the extracellular matrix on a molecular level.

Animals↗
PubMed1982

Halobacterium halobium phage øH.

Phage øH, a novel virus of the archaebacterium Halobacterium halobium, resembles in size and morphology two other Halobacterium phages. One-step growth curves show a 5.5 h eclipse, a latent period of 7 h, and an apparent burst size of 170. Phage øH contains linear, double-stranded DNA which has a molecular weight of 39 x 10 and a GC content of 65%. A packaging model accounting for the partial circular permutation and terminal redundancy of øH DNA is suggested. Partial homology of øH DNA with the DNA of H. halobium, predominantly with the AT-rich satellite DNA, was observed. The presence of minor restriction fragments of øH DNA which could be removed by purification of phage from single plaques suggests the existence of phage variants with rearranged DNA. A strain of H. halobium containing øH DNA was isolated which is resistant to infection by phage øH.

Journal Article↗