Absence Of Cell Surface Expression Research Articles

Absence of cell surface expression of human ACE leads to perinatal death.

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized most often by perinatal death. It is due to the inactivation of any of the major genes of the renin-angiotensin system (RAS), one of which is the angiotensin I-converting enzyme (ACE). ACE is present as a tissue-bound enzyme and circulates in plasma after its solubilization. In this report, we present the effect of different ACE mutations associated with RTD on ACE intracellular trafficking, secretion and enzymatic activity. One truncated mutant, R762X, responsible for neonatal death was found to be an enzymatically active, secreted form, not inserted in the plasma membrane. In contrast, another mutant, R1180P, was compatible with life after transient neonatal renal insufficiency. This mutant was located at the plasma membrane and rapidly secreted. These results highlight the importance of tissue-bound ACE versus circulating ACE and show that the total absence of cell surface expression of ACE is incompatible with life. In addition, two missense mutants (W594R and R828H) and two truncated mutants (Q1136X and G1145AX) were also studied. These mutants were neither inserted in the plasma membrane nor secreted. Finally, the structural implications of these ACE mutations were examined by molecular modelling, which suggested some important structural alterations such as disruption of intra-molecular non-covalent interactions (e.g. salt bridges).

Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria

Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

Mutations in the apically located Na(+)-K(+)-2Cl(-) co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the co-transporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues (1081)LLV(1083) as an ER exit signal necessary for maturation of NKCC2. Mutation of (1081)LLV(1083) to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with the LLV motif compromise co-transporter surface delivery through defective trafficking.

Functional consequence of a novel Y129C mutation in a patient with two contradictory melanocortin-2-receptor mutations

ContextFamilial glucocorticoid deficiency (FGD) is a rare autosomal recessive disease, characterised by isolated glucocorticoid deficiency in the absence of mineralocorticoid deficiency. Inactivating mutations in the ACTH receptor (melanocortin-2-receptor, MC2R) are well described and account for ∼25% of cases. By contrast, activating MC2R mutations are extremely rare.PatientWe report a child of Saudi Arabian origin who was diagnosed with FGD following hypoglycaemic episodes that resulted in spastic quadriplegia.Methods and resultsMC2R gene analysis revealed an unusual combination of two homozygous missense mutations, consisting of the novel mutation Y129C and the previously described F278C activating mutation. Parents were heterozygous at both of these sites. In vitro analysis of the Y129C mutation using a fluorescent cell surface assay showed that this mutant was unable to reach the cell surface in CHO cells stably transfected with MC2R accessory protein (MRAP), despite the demonstration of an interaction with MRAP by co-immunoprecipitation. The double mutant Y129C-F278C also failed to traffic to the cell surface.ConclusionThe tyrosine residue at position 129 in the second intracellular loop is critical in MC2R folding and/or trafficking to the cell surface. Furthermore, the absence of cell surface expression of MC2R would account for the lack of activation of the receptor due to the F278C mutation located at the C-terminal tail. We provide a novel molecular explanation for a child with two opposing mutations causing severe FGD.

An Essential Ligand-binding Domain in the Membrane Receptor for Retinol-binding Protein Revealed by Large-scale Mutagenesis and a Human Polymorphism

Plasma retinol-binding protein (RBP), the principal carrier of vitamin A in the blood, delivers vitamin A from liver, the site of storage, to distant organs that need vitamin A, such as the eye, brain, placenta, and testis. STRA6 is a high-affinity membrane receptor for RBP and mediates vitamin A uptake in these target organs. STRA6 is a 74-kDa multi-transmembrane domain protein that represents a new class of membrane transport protein. In this study, we used an unbiased strategy by analyzing >900 random mutants of STRA6 to study its structure and function, and we identified an essential RBP-binding domain in STRA6. Mutations in any of the three essential residues in this domain can almost completely abolish binding of STRA6 to RBP and its vitamin A uptake activity from holo-RBP without affecting its cell surface expression. We have also functionally characterized the mutations in human STRA6 that cause severe birth defects as well as several human polymorphisms. All STRA6 mutants associated with severe birth defects have largely abolished vitamin A uptake activity, consistent with the severe clinical phenotypes. In addition, we have identified a human polymorphism that significantly reduces the vitamin A uptake activity of STRA6. Interestingly, the residue affected by this polymorphism is located in the RBP-binding domain we identified, and the polymorphism causes decreased vitamin A uptake by reducing RBP binding. This study identifies an essential functional domain in STRA6 and a human polymorphism in this domain that leads to reduced vitamin A uptake activity.

Functional analysis of human decidual CD3-negative large granular leukocyte clones

OBJECTIVE: Atypical expression of human leukocyte antigen histocompatibility molecules or complement regulatory proteins by placental trophoblast has been hypothesized as a mechanism for spontaneous abortion. The purpose of this study was to determine expression of these proteins by placental villous trophoblast and to identify leukocyte populations within uteroplacental tissues from women with their first spontaneous abortion, their fourth or more recurrent spontaneous abortion, and from women having elective pregnancy termination.STUDY DESIGN: Fresh uteroplacental tissues were obtained at 6 to 9 weeks' gestation from eight women with their first spontaneous abortion, 20 women experiencing their fourth or more unexplained recurrent spontaneous abortion, and 20 women having an elective pregnancy termination. These tissues were analyzed immunohistochemically for human leukocyte antigen histocompatibility molecules (class I and II major histocompatibility complexes), complement regulatory proteins (CD46, CD55), and leukocyte phenotypes (CD45, CD3, CD14, CD56).RESULTS: There was absence of cell surface expression of class I and II human leukocyte antigen molecules but strong trophoblast expression of complement regulatory proteins in all villous placental samples. Leukocyte infiltration was noted in all decidual specimens. The predominant decidual leukocyte population was CD3-negative, CD56-positive cells, except in four cases of recurrent abortion where the normal ratio (≤2:3) of CD14-positive macrophages to CD56-positive leukocytes was >2:1. There was an unusual retention of maternal class II major histocompatibility complex-positive leukocytes within intervillous spaces attached to the apical surface of syncytiotrophoblast from one individual with recurrent abortion.CONCLUSION: Our data do not support the hypothesis that some cases of recurrent spontaneous abortion result from atypical expression of human leukocyte antigen histocompatibility molecules or lack of complement regulatory protein expression by placental villous trophoblast. These data suggest that occasional cases of recurrent spontaneous abortion could be associated with an impaired CD56-positive leukocyte response in the early decidualized endometrium.

The [formula omitted] gene is actively transcribed during all stages of murine B-lymphocyte development

It is generally accepted that the expression of FcϵRII CD23 on the surface of B-lineage cells is restricted to the stage of the resting, mature ( sIgM + sIgD + ) B-lymphocyte. However, it is unknown whether activation of the FcϵRII CD23 gene is also restricted to the stage of the mature B-lymphocyte. To address this question we investigated a panel of B-lineage cell lines for the presence of transcripts encoding FcϵRII CD23 . We detected transcripts in 16 of 26 B-lineage cell lines representing the entire spectrum of B-cell development. In most cases (13 of 16) active transcription of the murine FcϵRII CD23 gene was not coupled with the expression of cell surface FcϵRII CD23 protein. The stage-restricted pattern of cell surface FcϵRII CD23 expression did not hold for all murine B-cell lines. One post-switch B-cell line ( sIgM − sIgG + ) expressed FcϵRII CD23 on the cell surface and another could be induced with IL-4 and LPS to express surface FcϵRII CD23 . Transcription of the murine CD23 gene in the absence of cell surface expression of FcϵRII CD23 does not appear to simply be an aberrant feature of transformed B-cells since we found transcripts, but not surface expression, in some normal splenic and peritoneal B-lymphocytes. Our findings suggest that the potential for expression of FcϵRII CD23 may occur over a much broader developmental window of the B-lineage than previously suspected. Transcription of the FcϵRII CD23 gene, in the absence of detectable cell surface protein expression in B-lineage cell lines, and in sort-purified B-lymphocyte subpopulations, implies that in addition to regulatory mechanisms already known, murine CD23 is also regulated through post-transcriptional mechanisms that have not yet been characterized.

Characterization of a HTLV-I-infected cell line derived from a patient with adult T-cell leukemia with stable co-expression of CD4 and CD8

A long-term T-cell line, termed SP +, was developed from a human T-cell leukemia virus type I (HTLV-I)-infected patient with adult T-cell leukemia that is dependent on exogenous IL-2 for growth. The SP + expresses a full complimentation of HTLV-I-specifrc viral proteins, and contains replication competent viral particles. Restriction enzyme digestion followed by Southern blot analysis demonstrated the presence of a single integrated proviral copy and limiting dilution analysis confirmed the clonality of the cell line. Interestingly, phenotypically, the SP + cell line is CD2 +, CD3 + and coexpresses CD4 and CD8, yet lacks TCRαβ and TCRτδ expression. Further ontogenetic characterization of the SP + cell line demonstrated the lack of thymic T-cell precursor markers, including absence of cell surface expression of CD1, intracellular thymic terminal deoxynucleotidyl transferase (TdT) enzyme, as well as message expression for V(D)J recombinase activating gene-1 (RAG-1). Furthermore, the SP + cell did express the message for the CD3δ chain. Taken together, these data suggest that the SP + cell line resulted from HTLV-I infection of a mature CD4 +/CDB + lymphocyte. This cell line can be potentially useful as a model, both for regulation of cellular functions by HTLV-I and for immunologic functions of mature dual CD4/CD8 positive T-cells.

Immunohistochemical studies of human uteroplacental tissues from first-trimester spontaneous abortion

OBJECTIVE: Atypical expression of human leukocyte antigen histocompatibility molecules or complement regulatory proteins by placental trophoblast has been hypothesized as a mechanism for spontaneous abortion. The purpose of this study was to determine expression of these proteins by placental villous trophoblast and to identify leukocyte populations within uteroplacental tissues from women with their first spontaneous abortion, their fourth or more recurrent spontaneous abortion, and from women having elective pregnancy termination. STUDY DESIGN: Fresh uteroplacental tissues were obtained at 6 to 9 weeks' gestation from eight women with their first spontaneous abortion, 20 women experiencing their fourth or more unexplained recurrent spontaneous abortion, and 20 women having an elective pregnancy termination. These tissues were analyzed immunohistochemically for human leukocyte antigen histocompatibility molecules (class I and II major histocompatibility complexes), complement regulatory proteins (CD46, CD55), and leukocyte phenotypes (CD45, CD3, CD14, CD56). RESULTS: There was absence of cell surface expression of class I and II human leukocyte antigen molecules but strong trophoblast expression of complement regulatory proteins in all villous placental samples. Leukocyte infiltration was noted in all decidual specimens. The predominant decidual leukocyte population was CD3-negative, CD56-positive cells, except in four cases of recurrent abortion where the normal ratio (≤2:3) of CD14-positive macrophages to CD56-positive leukocytes was >2:1. There was an unusual retention of maternal class II major histocompatibility complex-positive leukocytes within intervillous spaces attached to the apical surface of syncytiotrophoblast from one individual with recurrent abortion. CONCLUSION: Our data do not support the hypothesis that some cases of recurrent spontaneous abortion result from atypical expression of human leukocyte antigen histocompatibility molecules or lack of complement regulatory protein expression by placental villous trophoblast. These data suggest that occasional cases of recurrent spontaneous abortion could be associated with an impaired CD56-positive leukocyte response in the early decidualized endometrium.

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.

The serological detection of recombinant vaccinia virus encoded HIV-1 gag proteins in the cytoplasm of human B cells in the absence of cell surface expression

Human lymphoblastoid B cells were acutely infected with recombinant vaccinia virus containing an HIV-1 gag gene (vac gag). Expression of HIV-1 gag proteins was measured in the cytoplasm, at the cell surface, and in the extracellular medium with monoclonal and human antibodies to HIV gag antigens. By indirect immunofluorescence staining and flow cytometric analysis HIV gag antigens were readily detected in the cytoplasm of cells within 4 hours of vac gag infection. However this was lost by 24 hours post-infection. In contrast, no specific staining was found at the cell surface after 4 or 24 hours. HIV gag antigen levels rose progressively in the extracellular medium during the first 24 hours after infection and showed an inverse correlation with cell viability. These results show that serologically detectable HIV core proteins are not expressed at the cell surface membrane in this system.