CD109 represents a novel branch of the alpha2-macroglobulin/complement gene family

We report here the genomic organization and phylogenic relationships of CD109, a member of the the alpha2-macroglobulin/complement (AMCOM) gene family. CD109 is a GPI-linked glycoprotein expressed on endothelial cells, platelets, activated T-cells, and a wide variety of tumors. We cloned full-length CD109 cDNA from the mammalian U373 cell line by RT-PCR and performed analysis of its corresponding genomic sequence. The CD109 cDNA spans 128 kb of chromosome 6q with its 33 exons constituting approximately 3.3% of the total CD109 genomic sequence. Sequence analysis revealed that CD109 contains specific motifs in its N-terminus, that are highly conserved in all AMCOM members. CD109 also shares motifs with certain other AMCOM members including: (1) a thioester 'GCGEQ" motif, (2) a furin site of four positively charged amino acids, and (3) a double tyrosine near the C-terminus. Based on a phylogenic analysis of human CD109 with other human homologs as well as orthologs from other mammalian species, C. elegans (ZK337.1) and E. coli homologs, we propose CD109 represents a novel and independent branch of the alpha2-macroglobulin/complement gene family (AMCOM) and may be its oldest member.     

Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein alpha2-macroglobulin

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.     

Limulus alpha 2-macroglobulin. First evidence in an invertebrate for a protein containing an internal thiol ester bond.

Intra-chain thiol ester bonds are present in a limited number of proteins. The thiol ester class of proteins includes vertebrate alpha 2-macroglobulin and the complement proteins C3 and C4. We report here the first instance of a thiol ester protein from an invertebrate, the alpha 2-macroglobulin proteinase-inhibitor homologue present in the plasma of the American horseshoe crab Limulus polyphemus. Our evidence is of three kinds: (1) the proteinase-binding activity of Limulus alpha 2-macroglobulin is inactivated by the low-molecular-mass primary amine methylamine; (2) the native protein is subject to autolytic fragmentation during mild thermal denaturation, yielding fragments of approx. 125 kDa and 55 kDa, whereas the methylamine-treated protein is stable under these conditions of thermal treatment; (3) new thiol groups are generated rapidly during reaction of the protein with trypsin. The demonstration of the thiol ester bond in a protein from an ancient invertebrate provides evolutionary evidence for the importance of this bond in the function of plasma forms of the alpha 2-macroglobulin-like proteinase inhibitors.     

Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.     

Identification of a gene in Leishmania infantum encoding a protein that contains a SP-RING/MIZ zinc finger domain

The SP-RING or Miz zinc finger domain that is related to the classical RING-finger motif, defines a class of proteins that can act as E3-like factors in the pathway of small ubiquitin-related modifier (SUMO) conjugation. This family includes the mammalian protein inhibitor of activated STAT (PIAS) proteins and related proteins from lower eukaryotes. Here we report the existence of a gene in Leishmania infantum, present as two identical copies placed upstream of each MAT2 gene copy, and transcribed as a single approximately 2.2 kb mRNA both in the logarithmic and stationary phases of the promastigote stage. This gene encodes a 47 kDa protein that has been named LORIEN. LORIEN is circumscribed to the cell periphery and it is antigenic during L. infantum infection of dogs and hamsters. Strikingly, this novel protein contains a highly conserved SP-RING/Miz zinc finger domain, raising the possibility that a SUMO or ubiquitin-like system may exist in this microorganism.     

A novel and highly conserved collagen (pro(alpha)1(XXVII)) with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family

The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro(alpha)1(XXVII) chain has a domain structure similar to that of the type B clade chains (alpha1(V), alpha3(V), alpha1(XI), and alpha2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro(alpha)1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240-110 kDa in tissue extracts and collagenous bands of 150-140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro(alpha) chains.     

Biosynthesis of rat alpha 1-macroglobulin. Identification of an intracellular precursor

Alpha 1-macroglobulin was purified from rat plasma by gel filtration (Sephacryl S-300) and ion exchange chromatography (DE52). Analysis of the purified alpha 1-macroglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides: a light chain which could be resolved into a double band (36/38 kDa) and a heavy chain (160 kDa). Under non-reducing conditions complexes of 200 and 400 kDa could be demonstrated. Antibodies were raised against both chains of alpha 1-macroglobulin which did not cross-react with either rat alpha 2-macroglobulin or rat alpha 1-inhibitor 3. It was shown that in the medium of [35S]methionine-labeled hepatocytes the two subunits of alpha 1-macroglobulin are linked by disulfide bridges. Intracellularly, however, a high molecular mass polypeptide (185 kDa) could be immunoprecipitated with either the antiserum to the heavy or the light chain of alpha 1-macroglobulin, indicating the existence of a polyprotein precursor. Also in a cell-free translation system alpha 1-macroglobulin was synthesized as a polyprotein consisting of heavy and light chains (162 kDa). In a pulse-chase experiment using tunicamycin to block N-glycosylation, alpha 1-macroglobulin secretion was totally inhibited. This finding reflects the importance of the oligosaccharide side chains for the proteolytic processing to the two subunits and/or secretion of alpha 1-macroglobulin.     

Identification and localization of a new human myotubularin-related protein gene, mtmr8, on 8p22-p23

Myotubularin and myotubularin-related proteins are dual-specificity phosphatases.Several myotubularin-related proteins have been identified in humans and mice. The members of the myotubularin protein family are highly conserved, from humans to yeast. Mutations in the human myotubularin gene (MTM1) lead to X-linked myotubular myopathy. Here we isolate and localize a novel putative myotubularin-related protein gene (MTMR8) on chromosome 8p22--p23,between the markers D8S550 and D8S265, by exon-trapping experiments and RT-PCR. Genomic sequencing revealed that the gene consists of 10 exons and spans approximately 43 kb. The corresponding cDNA is 7081 bp. The open reading frame predicts a protein of 549 amino acids and a calculated molecular mass of 63 kDa. Like myotubularin-related protein-5, MTMR8 has no dual-specificity phosphatase domain. It contains a double-helical motif similar to the SET interaction domain, which is thought to have a role in the control of cell proliferation.     

Genomic organization of the rat aspartyl-tRNA synthetase gene family: A single active gene and several retropseudogenes

The genomic organization of the gene encoding rat aspartyl-tRNA synthetase (AspRS), a class II aminoacyl-tRNA synthetase (aaRS), was determined. A single active gene and several pseudogenes were isolated from a rat genomic DNA library and characterized. The active DRS1 gene encoding the rat AspRS spans approximately 60 kb and is divided into 16 exons. Exons 8–16, encoding the nt-binding domain of the synthetase, are clustered in the 3′-region of the gene, whereas exons 3, 4, and 5, encoding the anticodon-binding domain are separated by large introns (up to 15 kb) containing LINE sequences. One of the pseudogenes, ΨDRSI, has a nt sequence 93% identical to that of the complete cDNA sequence of rat AspRS but several stop codons interrupt the coding sequence, thus identifying ΨDRS1 to an inactive processed pseudogene. Two repetitive elements from the LINE family are inserted into ΨDRS1. Calculation of nt substitution rates suggests that ΨDRS1 sequences arose approximately 27 Myr ago. The other pseudogene, ΨDRS2, should be more ancient. Taken together, these results clearly demonstrate that the AspRS gene family is composed of only one active gene. The availability of the gene structure of AspRS could help to clarify molecular evolution of class II aaRS.     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

Limited proteolysis of the alpha-macroglobulin rat alpha 1-inhibitor-3. Implications for a domain structure.

Rat alpha 1-inhibitor-3 is a 180-kDa monomeric proteinase inhibitor found in high concentration in rat plasma. By several criteria it has been shown to be a member of the family of alpha-macroglobulin proteinase inhibitors often exemplified by the tetrameric human alpha 2-macroglobulin. We have used limited proteolysis of rat alpha 1-inhibitor-3 to probe the domain structure of this family of proteins. Proteinases of different specificities, including trypsin, chymotrypsin, thermolysin, and Staphylococcus aureus V8 proteinase, were employed and a common fragmentation pattern was observed when the reaction products were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These fragments were electrotransferred to polyvinylidene difluoride membranes and subjected to NH2-terminal amino acid sequence analysis in order to position them within the context of the primary structure. The fragmentation pattern may define the domain structure of alpha 1-inhibitor-3 and serve as a model for the domain organization of the family of alpha-macroglobulin proteinase inhibitors.     

Binding, surface mobility, internalization, and degradation of rhodamine-labeled alpha 2-macroglobulin

We have used quantitative fluorescence methods to examine the fate of rhodamine-labeled alpha 2-macroglobulin (R-alpha 2 M) after binding to cell-surface receptors on NRK and Swiss 3T3 cells. From measurements of fluorescence intensities in NRK cells fixed after incubation with R-alpha 2M, we found that uptake was saturable and that half-maximal uptake occurred at 130 nM R-alpha 2M. Fluorescence measurements on cell extracts of NRK and Swiss 3T3 cells also showed a half-maximal uptake of R-alpha 2M near 130 nM. We estimate that NRK cells can take up 10(6) molecules of R-alpha 2M per hour via receptor-mediated endocytosis. The mobility of alpha 2-macroglobulin receptors on the surface of Swiss 3T3 cells was measured by using fluorescence photobleaching recovery. The two-dimensional effective diffusion coefficient of R-alpha 2M receptors was approximately 8 X 10(-10) cm2 s-1, a value close to that previously obtained for insulin and epidermal growth factor receptors. Degradation of R-alpha 2M by the cells was followed by using the loss of fluorescence from the 185000-dalton band in sodium dodecyl sulfate--polyacrylamide gels. Rhodamine fluorescence was detected in the gels by using a microscope fluorescence spectrophotometer. NRK cells degraded alpha 2M to low molecular weight fragments with a t 1/2 of 15 min. Swiss 3T3 cells degraded about 75% of the alpha 2M with a t 1/2 of 1 h. The remaining 25% remained as the intact 185000-dalton peptide after 24 h. No significant accumulation of large breakdown products was observed in Swiss 3T3 or NRK cells.     

Genomic definition of RIM proteins: evolutionary amplification of a family of synaptic regulatory proteins

RIMs are synaptic proteins that are essential for normal neurotransmitter release. We now show that while invertebrates contain only a single RIM gene, vertebrates contain four: two large genes encoding RIM1alpha (0.50 Mb) or RIM2alpha, 2beta, and 2gamma (0.50-0.75 Mb) and two smaller genes encoding RIM3gamma (14 kb) or RIM4gamma (55 kb). RIM1alpha and RIM2alpha consist of an N-terminal Zn(2+)-finger domain, central PDZ and C(2)A domains, and a C-terminal C(2)B domain; RIM2beta consists of a short beta-specific sequence followed by central PDZ and C(2)A domains and a C-terminal C(2)B domain; and RIM2gamma, 3gamma, and 4gamma consist of only a C(2)B domain. In the RIM2 gene, RIM2beta and 2gamma are transcribed from internal promoters. alpha- and beta-RIMs are extensively alternatively spliced at three canonical positions, resulting in >200 variants that differ by up to 400 residues. Thus gene duplication, alternative splicing, and multiple promoters diversify a single invertebrate RIM into a large vertebrate protein family. The multiplicity of vertebrate RIMs may serve to fine-tune neurotransmitter release beyond a fundamental, evolutionarily conserved, and common function for RIMs.     

Reaction of proteinases with alpha 2-macroglobulin from the American horseshoe crab, Limulus.

The products generated by the reaction of Limulus alpha 2-macroglobulin with trypsin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Unreacted Limulus alpha 2-macroglobulin had a subunit molecular mass of 185 kDa. Trypsin-reacted samples contained two prominent peptides smaller (85 and 100 kDa) and three peptides larger (200, 250, and 300-350 kDa) than the unreacted subunit. Reaction of methylamine-treated Limulus alpha 2-macroglobulin with trypsin resulted in the same two prominent reaction products smaller than 185 kDa, but all of the reaction products larger than 185 kDa were absent. The covalent binding of biotinylated trypsin with Limulus alpha 2-macroglobulin was detected by probing Western blots with horseradish peroxidase-avidin. Surprisingly, the only reaction products that contained trypsin were bands at 100 and 120 kDa. The staining of these bands with horseradish peroxidase-avidin was weak: most of the biotinylated trypsin that remained associated with alpha 2-macroglobulin during gel filtration chromatography was located at the dye front following reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products larger than 185 kDa did not contain trypsin. Methylamine-reacted Limulus alpha 2-macroglobulin failed to bind any biotinylated trypsin. In contrast to the reaction of trypsin with Limulus alpha 2-macroglobulin, all high molecular mass bands generated by the reaction of human alpha 2-macroglobulin with biotinylated trypsin stained intensely with horseradish peroxidase-avidin. Thus, Limulus alpha 2-macroglobulin forms thiol ester-dependent, high molecular mass products involving isopeptide bonding between trypsin-generated fragments, without the incorporation of trypsin into the complexes. Most of the alpha 2-macroglobulin-associated trypsin is non-covalently trapped rather than covalently cross-linked.     

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.