An isopeptide bond splitting enzyme from Hirudo medicinalis similar to gamma-glutamyl transpeptidase.

A new enzyme from Hirudo medicinalis capable of splitting gamma-glutamyl-p-nitroanilide and Glu--Lys-(N6-gamma-glutamyllysine) (isopeptidic bond between the epsilon-amino group of lysine and the gammacarboxylic group of glutamic acid) isopeptide bonds was purified. The protein was partially sequenced at the amino acid level, and the complete nucleotide and amino acid sequences were determined after cDNA cloning. The new enzyme has more than 60% similarity at the amino acid level to vertebrate gammaglutamyl transpeptidase (gamma-GT). According to the cDNA, the new protein has a molecular mass of 65 521 Da and a length of 600 amino acids.

Glycosylation of recombinant ancrod from Agkistrodon rhodostoma after expression in mouse epithelial cells.

The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two-dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-NAc antenna. Studies at the level of individual N-glycosylation sites demonstrated that glycans with N, N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.

A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus.

A thrombin-specific inhibitor with an apparent molecular mass of 11 kDa has been purified from the insect Rhodnius prolixus. Amino-terminal protein sequence analysis allowed the molecular cloning of the corresponding cDNA. The open reading frame codes for a protein of about 103 amino acid residues and displays an internal sequence homology of residues 6-48 with residues 57-101 indicating a two-domain structure. Based on the amino acid sequence the two domains exhibit high homology to protease inhibitors belonging to the Kazal-type family. Model building suggests that the first domain binds to the active site with residue His10 pointing into the specificity pocket. From gel filtration and tight-binding inhibition experiments the inhibitor appears to form 1:1 complexes with thrombin. Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhibitor. Natural and recombinant rhodniin both display inhibition constants of about 2 x 10(-13) M.

Isolation, sequence analysis, and cloning of haemadin. An anticoagulant peptide from the Indian leech.

A slow, tight-binding inhibitor of thrombin with an apparent molecular mass of about 5 kDa has been isolated from Haemadipsa sylvestris, an Indian leech of the family of Haemadipsidae. The inhibitory activity, called haemadin, is thrombin specific since it does not inhibit other proteases like trypsin, chymotrypsin, factor Xa, or plasmin. NH2-terminal amino acid sequence analysis (residues 1-45) does not reveal any homology to known serine protease inhibitors, including the thrombin-specific inhibitor hirudin. The haemadin cDNA cloned by polymerase chain reaction techniques codes for a polypeptide of 57 amino acid residues preceded by 20 residues of a signal peptide sequence. A synthetic gene coding for the mature haemadin was expressed in Escherichia coli. Recombinant haemadin displays a similar inhibition constant and specific activity as its natural counterpart. Although there is no obvious sequence identity between haemadin and hirudin, both proteins seem to share common mechanisms for thrombin inhibition.

Testis-specific beta 2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous beta 1 tubulin.

In Drosophila as in many organisms beta tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the beta 1 and beta 2 tubulins of Drosophila melanogaster and the beta 2 tubulin of D. hydei, and found these insect beta tubulins to be highly conserved and like beta tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. The beta 1 tubulin gene of Drosophila is constitutively expressed, whereas the beta 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the beta 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific beta 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent beta tubulins.

Cloning and identification of the gene coding for the 140-kd subunit of Drosophila RNA polymerase II.

Genomic clones of Drosophila melanogaster were isolated from a lambda library by cross-hybridization with the yeast gene coding for the 150-kd subunit of RNA polymerase II. Clones containing a region of approximately 2.0 kb with strong homology to the yeast gene were shown to code for a 3.9-kb poly(A)-RNA. Part of the coding region was cloned into an expression vector. A fusion protein was obtained which reacted with an antibody directed against RNA polymerase II of Drosophila. Peptide mapping of the fusion protein yielded a number of spots identical with spots derived from the 140-kd subunit of Drosophila RNA polymerase II. Sequence comparison of a segment of the Drosophila and the corresponding yeast clone yielded a high degree of homology at the protein level also, suggesting that we had isolated the gene coding for the 140-kd subunit of RNA polymerase II from Drosophila. In situ hybridization localized the DmRP140 gene at 88 A/B on chromosome 3 while the DmRP215 gene has previously been localized at 10 C on the X chromosome. Analysis of the transcripts (7.0 and 3.9 kb) in female and male flies shows dosage compensation for the transcription of the DmRP215 gene.

Characterization and developmental expression of beta tubulin genes in Drosophila melanogaster.

Genomic clones containing beta tubulin sequences were isolated from a lambda library of Drosophila melanogaster. In situ hybridization localized three genes to 56D and 60B on chromosome 2 as well as to 85D on chromosome 3. The latter was known through genetic analysis to be specifically expressed during spermatogenesis. The genomic clone, pTu85, derived from this region contains one complete beta tubulin coding region as well as the 3' end of an additional so far unidentified beta tubulin gene. Genomic Southern hybridizations reveal a total of five fragments with beta tubulin homology. Clone pTu56 codes for an RNA of 1.8 kb which is expressed in all developmental stages. Clone pTu60 codes for a 2.5-kb RNA expressed during embryogenesis and pupation. In testes RNA we detected a 2.2-kb message homologous to pTu85.

A DNA sequence of Drosophila melanogaster with a differential telomeric distribution.

A DNA sequence (8-19T) of 2.3 kilobase pairs (kb) of Drosophila melanogaster was localized by in situ hybridization to the extreme ends of polytene chromosomes and to the chromocenter. The relative abundance of this sequence at the ends of polytene chromosomes X:2L:2R:3L:3R is 1:3.4:1.9:0:2.7. This differential distribution is probably due to different copy numbers at the individual telomeric regions. Restriction enzyme analysis of genomic DNA shows that 8-19T sequences are interspersed with other sequences. The clone 8-19T, which contains most of this interspersed repetitive sequence, is itself not internally repetitive but has a complex sequence composition. Some of these sequences are transcribed into poly(A)+RNA. We suggest that the ends of Drosophila chromosomes are of a complex arrangement with some sequences common to all ends.