CDCP1 cleavage is necessary for homodimerization-induced migration of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks the estrogen, progesterone and HER2 receptors and is resistant to targeted and hormone therapies. TNBCs express high levels of the transmembrane glycoprotein, complement C1r/C1s, Uegf, Bmp1 (CUB)-domain containing protein 1 (CDCP1), which has been correlated with the aggressiveness and poor prognosis of multiple carcinomas. Full-length CDCP1 (flCDCP1) can be proteolytically cleaved, resulting in a cleaved membrane-bound isoform (cCDCP1). CDCP1 is phosphorylated by Src family kinases in its full-length and cleaved states, which is important for its pro-metastatic signaling. We observed that cCDCP1, compared with flCDCP1, induced a dramatic increase in phosphorylation of the migration-associated proteins: PKCδ, ERK1/2 and p38 mitogen-activated protein kinase in HEK 293T. In addition, only cCDCP1 induced migration of HEK 293T cells and rescued migration of the TNBC cell lines expressing short hairpin RNA against CDCP1. Importantly, we found that only cCDCP1 is capable of dimerization, which can be blocked by expression of the extracellular portion of cCDCP1 (ECC), indicating that dimerization occurs through CDCP1's ectodomain. We found that ECC inhibited phosphorylation of PKCδ and migration of TNBC cells in two-dimensional culture. Furthermore, ECC decreased cell invasiveness, inhibited proliferation and stimulated apoptosis of TNBC cells in three-dimensional culture, indicating that the cCDCP1 dimer is an important contributor to TNBC aggressiveness. These studies have important implications for the development of a therapeutic to block CDCP1 activity and TNBC metastasis.

Characterization of human tumor-infiltrating lymphocytes expanded in hollow-fiber bioreactors for immunotherapy of cancer.

We attempted to grow tumor-infiltrating lymphocytes (TIL) from 34 fresh tumors of eight different histologies using flasks for the initiation phase and hollow fiber bioreactors to expand TIL to therapeutic numbers. Overall success rate was 76% (26/34) including melanoma (9/14, 64%) and renal cell carcinoma (11/11, 100%). The mean number of days required to reach successful initiation (1 x 10(9) TIL) for all tumor types was 29 +/- 16 days (mean +/- S.D.). Therapeutic doses of TIL required an average of 88 +/- 23 days (initiation plus expansion) with an average TIL number of 3.2 x 10(10) +/- 2.8 x 10(10). TIL phenotype was predominantly CD4+ in 53% (16/30) and CD8+ in 47% (14/30), renal cell carcinoma samples accounted for 12/14 of the predominantly CD8+ TIL. Cells bearing the natural killer (NK) phenotype represented only 0-7% of TIL while LAK phenotype represented 0-68% (mean 11 +/- 15%); LAK was the predominant phenotype in one patient with kidney cancer. Cytotoxicity tests showed consistent NK and LAK activity in addition to cytolysis of autologous tumor. Autologous tumor cell restricted cytolysis was noted for three TIL cultures. The overall success rate and characteristics of TIL were similar to our results with TIL expanded in semi-permeable plastic bags. This work confirms that hollow-fiber bioreactors are a suitable alternative to semi-permeable bags and roller bottle systems for the expansion of human TIL for therapeutic use in cancer patients.

Degradation of stearoyl-coenzyme A desaturase: endoproteolytic cleavage by an integral membrane protease.

Stearoyl-coenzyme A desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a prototype for selective degradation of resident proteins of the endoplasmic reticulum. Using detergent-solubilized, desaturase-induced rat liver microsomes we have characterized a protease that degrades SCD. Degradation of SCD in vitro is highly selective, has a half-life of 3-4 h, and generates a 20-kDa C-terminal fragment of SCD. The N terminus of the 20-kDa fragment was identified as Phe177. The cleavage site occurs in a conserved 12-residue hydrophobic segment of SCD flanked by clusters of basic residues. The SCD protease remains associated with microsomal membranes after peripheral and lumenal proteins have been selectively removed. SCD protease is present in normal rat liver microsomes and cleaves purified SCD. We conclude that rapid turnover of SCD involves a constitutive microsomal protease with properties of an integral membrane protein.

Interleukin-2 induces N-glycosylation in T-cells: characterization of human lymphocyte oligosaccharyltransferase.

We have investigated the enzyme mediating N-glycosylation in "resting" and activated lymphocytes. Normal peripheral blood lymphocytes (PBLs) were found to have low activity for glycosylation of a synthetic glycan acceptor peptide. N-glycosylation activity increased 10-fold after mitogen activation of PBLs. N-glycosylation activity remained elevated during long-term culture and expansion of human lymphocytes when growth was supported by interleukin-2. To our knowledge, this is the first biochemical evidence for induction of endoplasmic reticulum functions during T-cell activation. The enzyme mediating N-glycosylation in lymphocytes was localized predominantly but not entirely to a microsomal organelle by subcellular fractionation. After solubilization and 85-fold purification from salt-washed microsomes, the enzyme preparation contained four predominant proteins. N-terminal sequence analysis identified the proteins as ribophorin I, ribophorin II (doublet), and a 50-kDa homologue of Wbp1, a yeast protein essential for N-glycosylation.

Isolation and structural analysis of microsomal membrane proteins.

Investigation of the endoplasmic reticulum requires a methodological background in the solubilization, purification, and structural analysis of membrane proteins. The experience of one laboratory with microsomal proteins over 30 years is summarized in this review. We focus on the isolation and structure of the major proteins of rabbit liver microsomes. The special pitfalls encountered with fragile protein complexes, hydrophobic peptides, and post-translational modifications are emphasized.

Purification and characterization of hepatic oligosaccharyltransferase.

Oligosaccharyltransferase transfers a preformed oligosaccharide from a dolichol carrier molecule to specific asparaginyl residues of proteins synthesized in the endoplasmic reticulum. We have isolated a protein complex with this activity from chicken liver microsomes with 850 fold purification. The purification procedure involved removal of peripheral and lumenal proteins, solubilization of the membranes by non-ionic detergent and glycerol gradient centrifugation. The complex was purified further by ion-exchange and gel filtration chromatography. SDS-PAGE analysis of the final preparation revealed 3 major protein bands, two bands with an approximate molecular weight of 65-kDa and one band of approximately 50-kDa. Endoglycosidase H digestion of the purified subunits indicated the presence of carbohydrate on the 65-I subunit. No carbohydrate was detected in the 65-II subunit or the 50-kDa subunit. Amino acid sequence analysis of the intact protein subunits and internal peptides generated by cynogen bromide digestion, identified the 65-kDa subunits as ribophorin I and II. The 50-kDa subunit has 25% homology with a yeast membrane protein (Wbplp) which is essential for oligosaccharyltransferase activity in Saccharomyces cerevisiae.

Human oligosaccharyltransferase: isolation, characterization, and the complete amino acid sequence of 50-kDa subunit.

Oligosaccharyltransferase (OT) catalyzes the glycosylation of asparagine residues in nascent polypeptides in the endoplasmic reticulum. In a previous communication we reported the purification and characterization of this enzyme from chicken oviduct. Here we describe the purification and sequence analysis of OT from human liver microsomes. Oligosaccharyltransferase copurified with three proteins designated 50-kDa, 65-I and 65-II based on their molecular weights by gel electrophoresis. The N-terminal sequence of the 50-kDa component was homologous to the 50-kDa subunit of avian OT. The N-terminal sequences of 65-I and 65-II were identical to the primary structures of human ribophorins I and II, respectively, predicted by cDNA sequencing. The complete amino acid sequence of the 50-kDa subunit of human OT was determined by chemical sequencing of peptides isolated from chemical and enzymatic digests. The 50-kDa subunit of human OT is 98% identical to its canine homolog, 93% identical to its avian homolog, and 25% identical to the beta subunit of yeast OT. These data indicate that structural features of oligosaccharyltransferase are conserved in all eukaryotes.

Microassay for oligosaccharyltransferase: separation of reaction components by partitioning in detergent solution followed by ultrafiltration.

Oligosaccharyltransferase catalyzes the transfer of oligosaccharide from a lipid dolichol to asparagine acceptor sites on nascent polypeptides. We have developed an assay for this enzyme which is based on the specific distribution of the substrate and product of the reaction in detergent solution. GlcNAc-[3H]GlcNAc-PP-Dol was synthesized for use as a carbohydrate donor. Benzoyl-Asn-Leu-Thr-amide, a commercially available peptide, was used as the oligosaccharyltransferase glycan acceptor substrate. In the presence of Triton X-100, GlcNAc-[3H]GlcNAc-PP-Dol partitions into detergent micelles while glycosylated acceptor peptide partitions into the intermicellar aqueous compartment. Separation of GlcNAc-[3H]GlcNAc-PP-Dol and glycopeptide was achieved by ultrafiltration. With this method oligosaccharyltransferase activity in microsomal preparations could be measured with as little as 1 microgram of protein.

Purification and characterization of avian oligosaccharyltransferase. Complete amino acid sequence of the 50-kDa subunit.

We have purified oligosaccharyltransferase from hen oviduct microsomes some 850-fold. Oligosaccharyltransferase activity copurified with a 200-kDa complex consisting of two 65-kDa polypeptides and a 50-kDa polypeptide. N-terminal sequence analysis indicated that the 50-kDa subunit was the avian form of OST48, a canine pancreatic microsomal protein associated with oligosaccharyltransferase. As the first step toward reconstitution of the oligosaccharyltransferase complex, the 50-kDa subunit was purified to homogeneity under nondenaturing conditions. The complete amino acid sequence of the 50-kDa subunit was determined by sequence analysis of peptides isolated by a combination of gel filtration and high performance liquid chromatography from chemical and enzymatic digests. The protein consists of 412 residues in a single polypeptide chain. The amino acid sequence of the 50-kDa subunit of avian oligosaccharyltransferase is 92% identical to the sequence of canine OST48 protein and about 25% identical to WBP1 protein from the yeast Saccharomyces cerevisiae. The yeast WBP1 protein has been shown in vitro, as well as in vivo, to be essential for the oligosaccharyltransferase activity.

Neural differentiation in small round cell tumors of bone and soft tissue with the translocation t(11;22)(q24;q12): an immunohistochemical study of 11 cases.

Clinical, cytogenetic, histopathologic, and immunohistochemical data were obtained in a series of 11 small round cell tumors (SRCT) of bone and soft tissue with the translocation t(11;22) (q24;q12). Ten cases were primary in bone, and one was of extraskeletal origin. According to conventional histopathologic criteria, 10 cases were Ewing's sarcomas (ES) and one was a peripheral neuroectodermal tumor (PNET). Besides the t(11;22), six cases had additional chromosomal aberrations, including trisomy 7 and partial trisomy for the long arm of chromosome 1, which have both been described as nonspecific secondary abnormalities often associated with tumor progression. The tumors were screened for neural differentiation with an antibody panel consisting of neuron-specific enolase, S100 protein, Leu-7, chromogranin, synaptophysin, and neurofilament. Three cases of ES were positive for S100 protein. The PNET and one case of ES were positive for neuron-specific enolase. All of the remaining immunohistochemical stains were negative. Hence, five of 11 SRCT of bone or soft tissue with the t(11;22) showed morphologic and/or immunohistochemical evidence of neural differentiation. In this limited series of cases, no cytogenetic or prognostic differences could be demonstrated between cases with and without a neural phenotype. Our results support the hypothesis that SRCT of bone of soft tissue with the t(11;22) form a single biologic entity displaying varying degrees of neuroectodermal differentiation. The clinical significance of additional cytogenetic abnormalities and of morphologic or immunohistochemical evidence of neural differentiation in this group of tumors needs to be further studied.

Calcium-dependent proteolysis of actin during storage of platelet concentrates.

In an ongoing study of the changes that occur in platelet concentrates during storage, we investigated two 28-26-Kd proteins designated SP-1 and SP-2, respectively, which increase markedly during blood-bank storage of platelet concentrates at room temperature. Formation of SP-1 and SP-2 was inhibited by storage at 4 degrees C as well as by treatment of the concentrates with leupeptin, N-ethylmaleimide, and EDTA; DFP and PPACK had no effect. The calcium ionophore A23187 markedly stimulated production of SP-1 and SP-2. After partial purification, the two proteins were found to be associated with platelet cytoskeletal protein. Two-dimensional peptide mapping and amino acid sequencing identified SP-1 and SP-2 as fragments of actin formed by cleavage on the N-terminal side of residues Thr-106 and Ala-114, respectively. Generation of SP-1 and SP-2 during storage of platelet concentrate is likely attributable to calcium-dependent neutral protease degradation of actin and may have implications for development of the platelet storage lesion.

Bilateral otitis media and hearing loss in an adult.

Bilateral otitis media, an uncommon entity in adults, may represent the initial manifestation of a life-threatening systemic disease. Prompt recognition and treatment of the underlying disease is needed to preserve auditory function and prevent involvement of other organ systems. We present the case of a thirty-four-year-old male with bilateral serous otitis media and progressive hearing loss, which was refractory to antimicrobial therapy and middle ear drainage. A mastoid biopsy revealed necrotizing granulomatous inflammation. The differential diagnosis and probable cause of this unusual finding are discussed.

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase.

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.

The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450.

The complete covalent structure of the constitutive cytochrome P-450, form 3b, from rabbit liver microsomes was determined. The apocytochrome contains 490 amino acid residues in a single polypeptide chain, Mr = 55,860. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. The constitutive form is only 46% homologous to the phenobarbital-induced cytochrome P-450 (Heinemann, F. S., and Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201) and contains a deletion at position 22. Strongly conserved regions include Cys435 and a previously identified tryptic peptide, residues 345-357. The distribution of hydrophobic segments is used to predict the membrane topology of the protein, and four possible orientations of this protein in the membrane are presented.

The covalent structure of hepatic microsomal epoxide hydrolase. I. Isolation and characterization of the cyanogen bromide fragments.

Microsomal epoxide hydrolase was purified to homogeneity from phenobarbital-induced rabbit liver for the purpose of determining the complete amino acid sequence. All of the expected 11 cyanogen bromide fragments of epoxide hydrolase were isolated by a combination of gel filtration and high pressure liquid chromatography. Each was characterized by its amino acid composition and NH2-terminal amino acid sequence. The complete amino acid sequences of the eight small fragments, from 5-29 residues, were determined.

The covalent structure of hepatic microsomal epoxide hydrolase. II. The complete amino acid sequence.

The complete amino acid sequence of hepatic microsomal epoxide hydrolase has been determined. The protein contains 455 amino acid residues in a single polypeptide chain and has Mr = 52,691. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. This is the first epoxide hydrolase and the third microsomal enzyme for which the complete sequence has been determined.

The complete amino acid sequence of rabbit phenobarbital-induced liver microsomal cytochrome P-450.

The complete amino acid sequence of the major phenobarbital-induced cytochrome P-450 (P-450LM2) from rabbit liver microsomes has been determined. The protein contains 489 amino acid residues in a single polypeptide chain and has Mr = 55,464. The sequence was compared with the amino acid sequence of P-450CAM and the nucleotide sequence of cDNA obtained from phenobarbital-induced rat liver cytochrome P-450 mRNA. These comparisons suggest that, despite functional similarities, the structural homology between microbial and microsomal cytochromes P-450 is limited to a single 8-residue region, and, in contrast, the structure of inducible microsomal cytochrome P-450 isozymes is highly conserved among mammalian species. Furthermore, we propose that the thiolate heme ligand of cytochrome P-450 is contributed by a cysteinyl residue near the COOH terminus, position 434 in the rabbit P-450LM2 sequence, based on the homology in this region with P-450CAM. The NH2 terminus of the protein from residues 1-310 is characterized by 8 hydrophobic segments 18-23 residues long, each of which is terminated by a cluster of charged amino acid residues. Residues 320-443 comprise a hydrophilic region which contains the putative heme binding cysteinyl residue as well as segments of homology with a constitutive rabbit cytochrome P-450 isozyme. The sequence data suggest that cytochrome P-450LM2 contains multiple transmembranous segments as well as a hydrophilic cytoplasmic domain. The hydrophilic domain contains regions of homology with several other cytochromes P-450, and thus appears to have an essential role in the biological function of the protein.

The covalent structure of rabbit phenobarbital-induced cytochrome P-450. Partial amino acid sequence and order of cyanogen bromide peptides.

The phenobarbital-induced isozyme of cytochrome P-450 was isolated from rabbit liver microsomes and subjected to chemical and proteolytic degradation. The resulting peptides were isolated exclusively by reverse phase high pressure liquid chromatography. Automated sequence analysis was employed to determine the linear arrangement of 385 residues and the order of all CNBr peptides. The NH2 terminus of the cytochrome P-450 consists of five hydrophobic segments 18 to 23 residues long, separated by polar regions of 8 to 15 residues. The COOH terminus contains a hydrophilic region 120 residues long as well as several hydrophobic segments. A tridecapeptide, previously found to be analogous to a tryptic peptide from a constitutive P-450 isozyme (Ozols, J., Heinemann, F. S., and Johnson, E. F. (1981) J. Biol. Chem. 256, 11405-11408) was positioned within the hydrophilic region of the COOH terminus. The amino acid sequence is interpreted in terms of a model for the topology of the cytochrome in the membrane which can be used as a basis for further experiments. Comparison of this sequence with the cytochrome P-450CAM sequence (Haniu, M., Armes, L. G., Tanaka, M., Yasunobo, K. T., Shastry, B. S., Wagner, G. C., and Gunsalus, I. C. (1982) Biochem. Biophys. Res. Commun. 105, 889-894) revealed a single homologous region which contains a cysteinyl residue. The amino acid sequence of his segment is as follows: P-450 microsomal/P-450CAM:Ile/Leu-Cys-Leu-Gly-Gln/Glu-Ser/Gly-Leu/Ile-Ala-Arg. The amino acid sequence data were also compared with the amino acid sequence deduced from the nucleotide sequence of phenobarbital-inducible cytochrome P-450 mRNA from rat liver (Kurijama-Fuji, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). This comparison suggests that the structure of the phenobarbital-induced isozyme is highly conserved among mammalian species.

Chemical structure of rat liver cytochrome b5. Isolation of peptides by high-pressure liquid chromatography.

The complete amino acid sequence of rat cytochrome b5 has been determined. Isolation of this species of cytochrome b5 in its native form from microsomes by means of detergent solubilization required the inclusion of the protease inhibitor, phenylmethylsulfonyl fluoride, throughout the isolation steps. Omission of the protease inhibitor yielded a 97-residue heme-containing peptide without the membranous segment. The primary structure of the intact molecules was deduced from automated sequence analysis of peptides generated by proteolytic or chemical cleavage and isolated exclusively by reversed-phase HPLC. The blocked amino terminus of cytochrome b5 was identified as N-acetylalanine. The hexosamine content of the cytochrome preparation was less than 0.1 mol/mol protein, indicating an absence of asparaginyl linked oligosaccharide.

Amino acid sequence of an analogous peptide from two forms of cytochrome P-450.

Two cytochrome P-450 preparations, a constitutive isozyme, form 3b, and a phenobarbital-induced isozyme, form 2, were isolated from rabbit liver microsomes and compared by peptide mapping following digestion with trypsin and by partial sequence analysis. The NH2-terminal sequence of form 3b differed from form 2 in 15 out of 18 amino acids, but both forms have an NH2-terminal methionine residue followed by an acidic residue. Comparisons of many of the tryptic peptides of the two forms by means of high pressure liquid chromatography, as well as amino acid composition and sequence analysis, indicated that peptides from these forms, with one exception, are different. A tridecapeptide, differing only in a methionine (form 3b)/isoleucine (form 2) replacement was isolated from both forms. The amino acid sequence of this peptide is as follows: Met-Pro-Tyr-Thr-Asp-Ala-Val-Ile/Met-His-Glu-Ile-Gln-Arg. Taken together, these data indicate that forms 2 and 3 represent dissimilar gene products. The observation that these two cytochromes share an analogous peptide suggests that this tridecapeptide may contribute structural information necessary for common functional properties.