The partial amino acid sequence of bovine cartilage proteoglycan, deduced from a cDNA clone, contains numerous Ser-Gly sequences arranged in homologous repeats.

We have determined the sequence of a partial cDNA clone encoding the C-terminal region of bovine cartilage aggregating proteoglycan core protein. The deduced amino acid sequence contains a cysteine-rich region which is homologous with chicken hepatic lectin. This lectin-homologous region has previously been identified in rat and chicken cartilage proteoglycan. The bovine sequence presented here is highly homologous with the rat and chicken amino acid sequences in this apparently globular region. A region containing clusters of Ser-Gly sequences is located N-terminal to the lectin homology domain. These Ser-Gly-rich segments are arranged in tandemly repeated, approx. 100-residue-long, homology domains. Each homology domain consists of an approx. 75-residue-long Ser-Gly-rich region separated by an approx. 25-residue-long segment lacking Ser-Gly dipeptides. These dipeptides are arranged in 10-residue-long segments in the 100-residue-long homology domains. The shorter homologous segments are tandemly repeated some six times in each 100-residue-long homology domain. Serine residues in these repeats are potential attachment sites for chondroitin sulphate chains.     

Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone.

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.     

Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetylgalactosamine residues of chondroitin. We previously reported the cloning of C4ST cDNA from mouse brain. We here report the cloning and expression of human C4ST cDNA. The cDNA was isolated from a human fetal brain cDNA library by hybridization with a DNA probe prepared from rat poly(A)(+) RNA used for the cloning of mouse C4ST cDNA. The cDNA comprises a single open reading frame that predicts a Type II transmembrane protein composed of 352 amino acids. The protein has an amino acid sequence homology of 96% with mouse C4ST. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that transfers sulfate to both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis indicated that human C4ST mRNAs (6.0 and 1.9 kb) are expressed ubiquitously in various adult human tissues. Dot blot analysis has shown that human C4ST is strongly expressed in colorectal adenocarcinoma and peripheral blood leukocytes, whereas strong expression of human chondroitin 6-sulfotransferase (C6ST) is observed in aorta and testis. These observations suggest that the expression of C4ST and C6ST may be controlled differently in human tissues. The C4ST gene was localized to chromosome 12q23.2-q23.3 by fluorescence in situ hybridization.     

Primary structure of human salivary alpha-amylase gene

A recombinant clone which covers the human salivary alpha-amylase gene in a single insert has been isolated from a human genomic DNA library using a human salivary alpha-amylase cDNA as a probe. Restriction mapping and nucleotide (nt) sequence analysis revealed that this gene is approx. 10 kb long and is separated into eleven exons by ten introns. Its 5'-flanking region has some sequence homology with that of mouse salivary alpha-amylase gene [Schibler et al., J. Mol. Biol. 155 (1982) 247-266].     

Genomic organization and expression pattern of mouse neuroglycan C in the cerebellar development

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan with an epidermal growth factor module that is expressed predominantly in the brain. Cloning studies with mouse NGC cDNA revealed the expression of three distinct isoforms (NGC-I, -II, and -III) in the brain and revealed that the major isoform showed 94. 3% homology with the rat counterpart. The NGC gene comprised six exons, was approximately 17 kilobases in size, and was assigned to mouse chromosome band 9F1 by fluorescence in situ hybridization. Western blot analysis demonstrated that, although NGC in the immature cerebellum existed in a proteoglycan form, most NGC in the mature cerebellum did not bear chondroitin sulfate chain(s), indicating that NGC is a typical part-time proteoglycan. Immunohistochemical studies showed that only the Purkinje cells were immunopositive in the cerebellum. In the immature Purkinje cells, NGC, probably the proteoglycan form, was immunolocalized to the soma and thick dendrites on which the climbing fibers formed synapses, not to the thin branches on which the parallel fibers formed synapses. This finding suggests the involvement of NGC in the differential adhesion and synaptogenesis of the climbing and parallel fibers with the Purkinje cell dendrites.     

Hyaluronate binding properties of versican.

We have previously cloned a large chondroitin sulfate proteoglycan (versican) from human fibroblasts. The primary sequence shows that the N terminus contains sequence homology with known hyaluronate-binding molecule, suggesting that versican can bind hyaluronate. To test this hypothesis we have reconstructed a full-length versican cDNA and a versican cDNA fragment encoding the N terminus and have transfected Chinese hamster ovary cells and mouse 3T3 fibroblasts, respectively, with these constructs. The transfected Chinese hamster ovary cells make a proteoglycan shown to be versican by enzymatic and immunologic analysis. No corresponding proteoglycan was seen in the control cells. Using hyaluronate affinity chromatography, we show that recombinant versican specifically binds hyaluronate and does not bind to heparin or chondroitin sulfate. The transfected fibroblasts make a 78-kDa truncated form of versican that also binds hyaluronate and does not bind the related polysaccharides, showing that the hyaluronate binding activity resides at the N terminus of versican. The binding of versican to hyaluronate is substrate-concentration dependent and time dependent and can be competed with unlabeled versican. The dissociation constant for versican binding to hyaluronate was determined to be 4 x 10(-9) M.     

The 66-kDa neurofilament protein (NF-66): Sequence analysis and evolution

A 2.5 kb cDNA clone encoding the mouse 66 kd neurofilament protein (NF-66) was isolated and sequenced. The deduced protein sequence contains 501 amino acid residues. Comparison of the mouse, rat and human NF-66 indicated >90% homology in protein sequence and 85% homology in coding nucleotide sequence. A high degree of homology was observed between NF-66 and other intermediate filament proteins especially in the α-helical domain. Zooblot analyses suggested that the putative ancestral gene for vimentin and NF-66 was detectable in the avian. By comparison, the ancestral sequence for GFAP appeared after that for vimentin. Special issue dedicated to Dr. Marion E. Smith.     

Cloning of human heparan sulfate proteoglycan core protein, assignment of the gene (HSPG2) to 1p36.1----p35 and identification of a BamHI restriction fragment length polymorphism.

We have isolated a cDNA coding for the core protein of the large basement membrane heparan sulfate proteoglycan (HSPG) from a human fibrosarcoma cell (HT1080) library. The library was screened with a mouse cDNA probe and one clone obtained, with a 1.5-kb insert, was isolated and sequenced. The sequence contained an open reading frame coding for 507 amino acid residues with a 84% identity to the corresponding mouse sequence. This amino acid sequence contained several cysteine-rich internal repeats similar to those found in component chains of laminin. The HSPG cDNA clone was used to assign the gene (HSPG2) to the p36.1----p35 region of chromosome 1 using both somatic cell hybrid and in situ hybridization. In the study of the polymorphisms of the locus, a BamHI restriction fragment length polymorphism was identified in the gene. This polymorphism displayed bands of 23 and 12 kb with allele frequencies of 76 and 24%, respectively.     

Molecular cloning and sequence analysis of the mouse kallikrein-binding protein gene.

A genomic clone (MKBP-10) encoding the mouse kallikrein-binding protein (MKBP) was isolated from a mouse genomic DNA library by screening with a rat kallikrein-binding protein (RKBP) cDNA probe. The total sequenced region of the MKBP gene spans 8615 base pairs. The exon and intron locations of the RKBP gene were identified by similarity with the RKBP gene. The MKBP gene encodes a prepeptide of 417 amino acid residues which exhibits 71% homology with RKBP. A TATA box sequence was located in the 5' flanking region of the MKBP gene by similarity with the consensus sequence TATAAAA.     

The primary structure of NG2, a novel membrane-spanning proteoglycan

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.     

Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.     

Human cellular retinol-binding protein gene organization and chromosomal location

The gene encoding the human cellular retinol-binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11-3qter on human chromosome 3 using hybridizations to mouse-human, rat-human and hamster-human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty-acid-binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the 'housekeeping' genes in that it is (G + C)-rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9-bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5' upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.