Selective Endocytosis Research Articles

Cancer-targeted polymeric drugs.

A major challenge in cancer chemotherapy is the selective delivery of small molecule anti cancer agents to tumor cells. Water-soluble polymer-drug conjugates exhibit good water solubility, increased half-life, and potent anti tumor effects. By localizing the drug at the desired site of action, macromolecular therapeutics have improved efficacy and enhanced safety at lower doses. Since small molecule drugs and macromolecular drugs enter cells by different pathways, multi-drug resistance (MDR) can be minimized. Anti-cancer polymer-drug conjugates can be divided into two targeting modalities: passive and active. Tumor tissues have anatomic characteristics that differ from normal tissues. Macromolecules penetrate and accumulate preferentially in tumors relative to normal tissues, leading to extended pharmacological effects. This "enhanced permeability and retention" (EPR) effect is the principal reason for current successes with macromolecular anti-cancer drugs. Both natural and synthetic polymers have been used as drug carriers, and several bioconjugates have been clinically approved or are in human clinical trials. While clinically useful anti-tumor activity has been achieved using passive macromolecular drug delivery systems, further selectivity is possible by active targeting. Attachment of targeting moieties to the polymer backbone can further exploit differences between cancer and normal cells through selective receptor-mediated endocytosis. This strategy would augment the EPR effect, thereby further improving the therapeutic index of the macromolecular drug. This review discusses the development and therapeutic potential of prototype macromolecular drugs for use in cancer chemotherapy. Specific examples are selected to illustrate the basic design principles for soluble polymeric drug delivery systems.

Lipid microdomains are required sites for the selective endocytosis and nuclear translocation of IFN-gamma, its receptor chain IFN-gamma receptor-1, and the phosphorylation and nuclear translocation of STAT1alpha.

IFN-gamma contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1alpha via a complex of IFN-gamma/IFN-gamma receptor (IFNGR)-1/STAT1alpha with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1alpha. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-gamma resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-gamma and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1alpha, but did not affect the binding of IFN-gamma to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1alpha were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-gamma, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-gamma binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1alpha or the nuclear translocation of IFN-gamma or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-gamma-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-gamma, IFNGR-1, and STAT1alpha.

Low-density Lipoprotein Receptor-related Protein Mediates the Endocytosis of Anionic Liposomes in Neurons

We have recently demonstrated that anionic liposomes efficiently introduce foreign DNA into postmitotic neurons and other cell types (Lakkaraju, A., Dubinsky, J. M., Low, W. C., and Rahman, Y.-E. (2001) J. Biol. Chem. 276, 32000-32007). To investigate the mechanism of liposome uptake, we followed the internalization of anionic liposome-encapsulated Cy3-labeled oligonucleotides (AL-Cy3ONs) by hippocampal neurons using confocal microscopy. Uptake of AL-Cy3ONs was widespread and time- and temperature-dependent, indicative of receptor-mediated endocytosis. The low-density lipoprotein receptor-related protein (LRP) was crucial for anionic liposome endocytosis because the receptor-associated protein or an anti-LRP antibody inhibited internalization, and fibroblasts lacking LRP did not internalize AL-Cy3ONs. Using selective endocytosis inhibitors, we found that liposome endocytosis and intracellular transport required clathrin, dynamin, an intact cytoskeletal network, and phosphatidylinositol 3-kinase activity. Cy3ONs did not significantly colocalize with recycling endosomal/lysosomal markers and entered neuronal nuclei within 1-3 h of incubation. Approximately 50% of the internalized liposomal phospholipids were recycled back to the cell surface, in keeping with the fluidity of their acyl chains. Liposome endocytosis did not require heparan sulfate proteoglycans or cause calcium influx into neurons. Thus, constitutive endocytosis of anionic liposomes by LRP utilizes only one component, in contrast to the more involved heparan sulfate proteoglycan-LRP pathway implicated in the pathogenesis of Alzheimer's disease.

Functional Organization of MIR2, a Novel Viral Regulator of Selective Endocytosis

Kaposi's sarcoma-associated herpesvirus encodes two related proteins, MIR1 and MIR2, that lead to reduction of the cell surface levels of major histocompatibility complex class I and other polypeptides involved in immune recognition. MIR1 and MIR2 do not affect the assembly or transport of their target proteins through the secretory pathway; rather, they act to enhance the selective endocytosis of target chains from the cell surface. Sequence inspection reveals that the modulator of immune recognition (MIR) proteins contain an NH(2)-terminal zinc finger of the plant homeodomain (PHD) subfamily, two transmembrane (TM) domains, and a C-terminal conserved region (CR). Here we examine the transmembrane topology and functional organization of MIR2. Both the PHD domain and the CR are disposed cytosolically and are essential for MIR-mediated endocytosis. MIR proteins form homo-oligomers; this activity is independent of the PHD and CR elements and maps instead to the TM regions. Analysis of chimeras between MIR1 and MIR2 reveals that the TM regions also mediate target selectivity. Mutations that ablate the PHD or CR regions generate dominant negative phenotypes for major histocompatibility complex class I endocytosis. These findings suggest a domain organization for the MIR proteins, with the TM regions involved in target selection and the cytosolic PHD and CR domains involved in the possible recruitment of cellular machinery that directly or indirectly regulates internalization of target molecules.

Regulation of cholangiocyte secretion.

Observations from a variety of model systems suggest that ductular bile formation is mediated in large part by transepithelial transport of Cl(-) ions and have identified Cl(-) channels in the apical membrane as important targets for hormones and other factors that modulate bile volume and composition through effects on duct cells. Signaling through secretin receptors that stimulate adenylyl cyclase and activate the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels represents a prototype for cholangiocyte secretion. However, recent observations indicate that cholangiocytes also express a variety of receptors that modulate secretory responses in the absence of effects on cyclic adenosine monophosphate (cAMP), and Cl(-) channels unrelated to CFTR have been identified. Moreover, rapid exocytosis of subapical vesicles is coupled closely to different Cl(-) secretory responses. These observations suggest that the protein composition of the apical domain is regulated by selective exocytosis and endocytosis of channel-containing vesicles, leading to rapid modulation of the transport capacity of individual cells in response to changing physiologic demands.

Differential Nuclear Localization of the IFNGR-1 and IFNGR-2 Subunits of the IFN-γ Receptor Complex Following Activation by IFN-γ

We have recently identified a nuclear localization sequence (NLS) in the C-terminus of murine type II interferon (IFN), IFN-gamma, that is responsible for the internalization and nuclear translocation of extracellularly added IFN-gamma. Because the uptake of IFN-gamma is a receptor-mediated endocytotic process, we examined in this study the fate of both the receptor subunits (IFNGR-1 and IFNGR-2) of the heterodimeric IFN-gamma receptor complex. Human IFN-gamma (HuIFN-gamma) was also found to contain a polybasic NLS in a conserved C-terminal region capable of directing its nuclear translocation. Like the ligand, the IFNGR-1 subunit of the receptor complex on WISH cells was found to be translocated to the nucleus on treatment with HuIFN-gamma. Using a combination of immunoprecipitation and immunofluorescence techniques, we found the nuclear accumulation of IFNGR-1 to be ligand dependent, and it was evident within 10-20 min after ligand stimulation. IFNGR-1 was found to colocalize, in a time-dependent and dose-dependent fashion, with the nuclear translocation of the transcription factor Stat1alpha, which is activated by this ligand-receptor system. In addition, Stat1alpha was found to be complexed with IFNGR-1 over the time period of its nuclear translocation. In marked contrast, IFNGR-2 was not transported to the nucleus. The surface immunofluorescence pattern of IFNGR-2 suggested that, following ligand stimulation, the majority of IFNGR-2 remains at the cell surface, whereas IFNGR-1 is endocytosed and targeted to the cell nucleus. These findings suggest that IFNGR-1 plays an active intracellular role in signal transduction events subsequent to the binding of ligand to the dimeric receptor complex. Furthermore, these studies provide the first example of the selective endocytosis and nuclear translocation of a subunit of a multimeric receptor complex.

Selective endocytosis of thyroglobulin: A review of potential mechanisms for protecting newly synthesized molecules from premature degradation

In 1976 Cortese, Schneider and Salvatore (Eur. J. Biochem. 68 (1976) 121–129) showed that the thyroid gland protects newly synthesized, iodine and hormone poor thyroglobulin from immediate degradation. Since then there has been substantial progress in understanding the mechanism by which this selectivity of degradation occurs. Thyroglobulin in the follicular lumen is internalized mainly by receptor-specific endocytosis. Recycling of immature, poorly iodinated thyroglobulin back to the follicular lumen is the pathway most likely responsible for selectivity. Since additional carbohydrate groups are added to the immature thyroglobulin, it appears that this recycling occurs via the Golgi compartment. The molecular signal for recycling most likely involves the complex carbohydrates and probably is exposed GlcNAc groups. A thyroid-specific GlcNAc receptor has been identified and cloned. Other Tg-binding sites have been identified in the thyroid, but their physiological role remains to be determined.

A Novel Mechanism of CD4 Down-modulation Induced by Monosialoganglioside GM3: INVOLVEMENT OF SERINE PHOSPHORYLATION AND PROTEIN KINASE C δ TRANSLOCATION

In this report the molecular mechanism(s) involved in the rapid and selective endocytosis of cell surface glycoprotein CD4 induced by exogenous monosialoganglioside GM3 in human peripheral blood lymphocytes have been investigated. Inhibition of the GM3-induced CD4 down-modulation was observed in the presence of specific protein kinase C (PKC) inhibitors. Scanning confocal microscopy revealed the translocation and clustering on the cell surface of PKC isozymes delta and theta (more evidently than alpha and beta) after GM3 treatment, suggesting the involvement of these isozymes in the ganglioside-induced CD4 down-modulation. Exogenous GM3 induced phosphorylation of CD4 molecule, which then dissociated from p56(lck), as early as after 5 min. Moreover, addition of GM3 resulted in a rapid (1 min) cytosolic phospholipase A2 activation with consequent arachidonic acid release, whereas no phosphatidylinositol-phospholipase C activity was observed. Both PKC translocation and CD4 down-modulation were blocked by the trifluoromethylketone analog of arachidonic acid, a selective inhibitor of cytosolic phospholipase A2 and by mitogen-activated protein kinase inhibitor PD98059. Taken together, these findings strongly suggest that GM3 may trigger a novel mechanism of modulation of the CD4 surface expression through the activation of enzyme(s) involved in the regulation of cellular functions.

Selective endocytosis of fluorothymidine and azidothymidine coupled to LDL into HIV infected mononuclear cells

Drug targeting via lipoproteins may be of benefit for use of cytotoxic drugs like fluorothymidine (FLT) or azidothymidine (AZT). Both drugs are potent inhibitors of the human immunodeficiency virus (HIV) reverse transcriptase and are used in the therapy of HIV infection. With regard to this project, the selective endocytosis in HIV infected human macrophages was studied after covalent coupling of AZT and LDL to low density lipoproteins (LDL). Cultured human macrophages and the lymphocytic Molt 4 8 cell line were infected with HIV-1 in vitro and subsequently treated with FLT-LDL or AZT-LDL. Viral replication was followed by determination of cell-released capsid antigen p24. Internalisation into HIV-1 infected human macrophages by the scavenger receptor pathway leads to a dose dependent inhibition of HIV replication. Otherwise, in HIV infected, but scavenger receptor missing lymphocytes (Molt 4 8 cells), neither endocytosis nor inhibition of HIV replication results. Thus, covalent coupling of drugs to LDL leads to a macrophage specific transport. This strategy could possibly avoid toxic side effects in the therapeutic use of antiretroviral drugs and thus may open a way for an earlier chemotherapy in HIV infection.

Moving Membrane up to the Front of Migrating Cells

Moving Membrane up to the Front of Migrating Cells

Endocytosis of albumin and thyroglobulin at the basolateral membrane of thyrocytes organized in follicles.

Serum proteins such as albumin are present inside thyroid follicles in both normal and pathological situations. To analyze the mechanism of entry of these proteins, we investigated the ability of polarized thyrocytes to internalize soluble molecules at their basolateral pole. Experiments were conducted on in vitro reconstituted thyroid follicles using BSA and pig thyroglobulin (Tg) coupled to gold particles for electron microscopy, conjugated to fluorescein for conventional and confocal fluorescence microscopy, or radioiodinated for biochemical measurements. Incubations were carried out at 37 C. BSA and Tg coupled to gold particles were rapidly internalized from the culture medium and sequentially found in small vesicles and early endosomes and in late endosomes and lysosomes. Fluorescence microscope analyses revealed that the majority of cells forming reconstituted thyroid follicles are capable of internalizing BSA and Tg, but that Tg was more efficiently endocytosed than BSA. Using radioiodinated ligands, it was observed that the endocytosis of Tg was 10 times higher than that of BSA. The internalization of [125I]Tg was inhibited by increasing concentrations of unlabeled Tg. In contrast, endocytosis of 125I-labeled BSA was independent of the unlabeled BSA concentration. Experiments performed at 4 C indicated the presence of a basolateral membrane binding activity for [125I]Tg; the Tg concentration that reduced the binding of labeled Tg by 50% ranged from 4-6 microM. These data are evidence of a process of internalization of soluble molecules at the basolateral pole of thyrocytes, with BSA being internalized by fluid phase endocytosis and Tg by selective endocytosis. Our findings explain how serum albumin can enter thyroid follicles and disclose a new cellular handling and transport pathway of Tg. We propose that selective uptake of Tg operating on molecules secreted at the basolateral surface of thyrocytes could control the amount of Tg released in the circulation.

Coupling of the antiviral drug ara-AMP to lactosaminated albumin leads to specific uptake in rat and human hepatocytes

We covalently coupled 9-β-D-arabinofuranosyladenine 5′-monophosphate (ara-AMP) to the carrier molecule lactosaminated human serum albumin using a water-soluble carbodiimide with a two-step conjugation method (pH 4.5 and pH 7.5) instead of the commonly used single-step conjugation at pH 7.5. This resulted in a predominantly monomeric conjugate (lac27-HSA–ara-AMP9). The conjugate was stable in buffer (pH 7.4) and blood plasma. After in vivo injection, the carrier and the monomeric conjugate were subjected to selective endocytosis in rat hepatocytes, as shown on immunohistochemical study and cellseparation techniques using 125I-labeled material. In competition experiments with other ligands for the asialoglycoprotein receptor N-acetylgalactosamine and asialofetuin, we showed that both lactosaminated human serum albumin and lac27-HSA–ara-AMP9 are subject to endocytosis by this receptor system. Although the coupling of ara-AMP significantly increased the net negative charge of the conjugate compared with the native carrier, liver uptake was not affected by coadministration of an excess of succinylated human serum albumin (suc-HSA), a negatively charged ligand for the scavenger receptor. Incubation studies with purified rat liver lysosomes showed that in this acidic and proteolytic environment, mainly ara-AMP and, to a much lesser extent, ara-A itself were released from the carrier. After injection into the rat in vivo and in isolated perfused rat liver, no free ara-AMP or 9-B-D-arabinofuranosyladenine (ara-A) could be detected in plasma and perfusate, respectively, indicating proper retention of the virally active components in hepatocytes. Uptake experiments with freshly isolated rat and human hepatocytes indicated that human hepatocytes can also carry out endocytosis of lactosaminated human serum albumin and the ara-AMP conjugate by means of the galactose-recognizing receptor. (HEPATOLOGY 1993;18:146–152).

Coupling of the antiviral drug ara-AMP to lactosaminated albumin leads to specific uptake in rat and human hepatocytes

We covalently coupled 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) to the carrier molecule lactosaminated human serum albumin using a water-soluble carbodiimide with a two-step conjugation method (pH 4.5 and pH 7.5) instead of the commonly used single-step conjugation at pH 7.5. This resulted in a predominantly monomeric conjugate (lac27-HSA-ara-AMP9). The conjugate was stable in buffer (pH 7.4) and blood plasma. After in vivo injection, the carrier and the monomeric conjugate were subjected to selective endocytosis in rat hepatocytes, as shown on immunohistochemical study and cell-separation techniques using 125I-labeled material. In competition experiments with other ligands for the asialoglycoprotein receptor N-acetylgalactosamine and asialofetuin, we showed that both lactosaminated human serum albumin and lac27-HSA-ara-AMP9 are subject to endocytosis by this receptor system. Although the coupling of ara-AMP significantly increased the net negative charge of the conjugate compared with the native carrier, liver uptake was not affected by coadministration of an excess of succinylated human serum albumin (suc-HSA), a negatively charged ligand for the scavenger receptor. Incubation studies with purified rat liver lysosomes showed that in this acidic and proteolytic environment, mainly ara-AMP and, to a much lesser extent, ara-A itself were released from the carrier. After injection into the rat in vivo and in isolated perfused rat liver, no free ara-AMP or 9-B-D-arabinofuranosyladenine (ara-A) could be detected in plasma and perfusate, respectively, indicating proper retention of the virally active components in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of yolk proteins in insect oocytes.

In insects, accumulation of yolk, or vitellogenesis, is a heterosynthetic pro­ cess, in which an extraovarian tissue, the fat body, produces yolk protein precursors ( 1 , 92). Heterosynthetic vitellogenesis occurs even in the most primitive insects, the Apterygota (9, 10, 1 13). Only for two apterygote insects have ultrastructural observations indicated participation of autosynthesis in vitellogensis (9, 10) . This claim, however, requires confirmation by biochem­ ical or immunochemical means . Because of the heterosynthetic nature of vitellogenesis in insects, their oocytes are highly specialized for the specific accumulation of extraovarian yolk protein precursors . Telfer first implicated the role of endocytosis in yolk deposition in insect oocytes . He found selective concentration of some hemolymph-borne proteins in yolk bodies of Hyalophora cecropia oocytes ( 1 29, 1 3 1 ) . Coated vesicles, the cellular structures associated with selective endocytosis, were first observed in oocytes of the mosquito Aedes aegypti ( 1 1 2) . These pioneering studies were followed by numerous electron micro­ scopical and tracer investigations that supported earlier data on the in­ ternalization pathway of extraovarian proteins in follicles and on the role of coated vesicles in yolk deposition in insect oocytes (reviewed in 1 33) .

Selective endocytosis of vitellogenin by oocytes of the mosquito, Aedes aegypti: An in vitro study

An in vitro assay was used to study the uptake of vitellogenin (VG) by oocytes of the mosquito, Aedes aegypti. The VG, obtained from the culture of fat bodies in vitro, was metabolically labeled to a high specific activity ( 3–6 × 10 5 cpm/μg) using [ 35S]methionine. Purification of both VG and vitellin (VN) from ovaries was achieved with ion-exchange chromatography on DEAE-Sepharose CL-6B. In an optimized in vitro system, mosquito oocytes in intact ovaries maintained stable endocytotic activity for at least 4 h. The rate of VG uptake was maximal at pH 7.5. It declined at acid pH and ceased abruptly at pH 6.3. Accumulation of VG by mosquito oocytes demonstrated features of receptor-mediated endocytosis; i.e. temperature dependence, saturability, selectivity and tissue specificity. The uptake of VG was inhibited at 4°C with only 6% of uptake achieved at 27°C. At 24 h post-blood meal, uptake of VG by oocytes at 27°C, neared saturation with a VG concentration in the medium of 8 μg/μl. Saturation kinetics generated for VG endocytosis by these oocytes produced a V max = 3.2 μg/μl/h and an apparent K uptake = 8.4 × 10 −6 M. Ovaries accumulated 10 times the amount of VG compared to mouse IgG, while uptake of VN was 71.6% of that for VG, indicating that oocytes from Aedes are able to distinguish these proteins. Non-ovarian tissues, fat body and Malpighian tubules, accumulated both VG and IgG at equally low levels. During the vitellogenic cycle, the rate of VG uptake by oocytes showed a steep and linear increase between 6 and 24 h post-blood meal. The peak of VG uptake occurred between 24 and 30 h post-blood meal, followed by a precipitous decline and cessation of uptake by 36 h.

Microglia in the neurohypophysis associate with and endocytose terminal portions of neurosecretory neurons

The rat neurohypophysis contains a population of microglial cells, the majority of which occupy a pericapillary position in the resting gland. The microglia are immunocytochemically identifiable by the presence of macrophage-associated antigens and resemble microglia of the CNS. Morphometry at light and electron microscopic levels reveals that such cells constitute approximately 19% of the intrinsic cell population, excluding the endothelial cells. Two other populations of neurohypophysial glial cells, parenchymatous pituicytes and fibrous pituicytes, do not express macrophage-associated antigens. The microglia have long processes which surround and, in some cases, engulf apparently viable portions of the magnocellular neurosecretory nerve terminals. A sequence of stages of selective endocytosis and degradation of the engulfed nerve terminals can be visualized within pericapillary microglia. Some phagosomes and secondary lysosomes contain morphologically intact neurosecretory granules; others contain partially destroyed neurosecretory granules or amorphous material all of which are identifiable as originating from the magnocellular neurosecretory terminals by their immunoreactivity for oxytocinor vasopressin-neurophysin. This finding indicates a novel role for the microglial cells in remodelling terminal arborizations of neurosecretory neurons and in processing or degrading hormones and peptides they contain. Because of their close and selective associations with other cellular elements of the neurohypophysis, any substances produced by microglia also have the potential to influence hormone secretion, pituicyte proliferation and neurohypophysial vasculature.

Selective endocytosis, in vitro, by ovarian follicles from Hyalophora cecropia

Ovarian follicles of Hyalophora cecropia, incubated in vitro with isolated and radiolabelled hemolymph and yolk proteins, provided a satisfactory model of in situ vitellogenesis. Uptake of proteins was specific. The follicles accumulated vitellogenin and microvitellin at constant rates for 6 hr, depositing them in the protein yolk spheres of the oocyte. Uptake of these two proteins was saturable by high concentrations of homologous protein and inhibited by p-dinitrophenol. In contrast, two other abundant hemolymph proteins, arylphorin and flavoprotein, were taken up at lower rates, and become concentrated primarily in the basement lamina of the follicle. Their accumulation was not saturable and not inhibited by p-dinitrophenol. The two yolk precursors were accumulated only by follicles at stages known to be vitellogenic, and the rates of uptake were shown to approximate the rates of accumulation of these proteins in situ. The uptake of vitellogenin, but not microvitellin, was enhanced 2- to 3-fold by hemolymph ultrafiltrates. Vitellin from mature eggs was not distinguishable from vitellogenin by the endocytotic apparatus. Finally, endocytotic uptake was not affected by inhibition of protein synthesis. This finding supports the concept of membrane and receptor recycling in yolk formation, and argues against an essential role of the follicle cell product paravitellogenin in the mechanism of hemolymph protein uptake.

In vitro study of selective endocytosis of vitellogenin by locust oocytes

Oocytes ofLocusta migratoria selectively sequester the female-specific protein vitellogenin at a high rate. In vitro studies using in vivo radiolabelled vitellogenin show that uptake rates increase proportionally with time and temperature (with a sharp change between 10° and 15°C). They also depend on oocyte size and vitellogenin concentration. The uptake process is saturable. Vitellogenin is taken up more specifically than vitellin. Bovine serum albumin, casein and male locust haemolymph proteins do not inhibit endocytosis of vitellogenin. However, several metabolic inhibitors and phosphorylated compounds, such as phosvitin or phosphohexoses, are potent inhibitors of vitelogenin incorporation.

766 CYSTINOTIC FIBROBLASTS ACCUMULATE CYSTINE FROM CYSTINE BUT NOT CYSTEINE RESIDUES OF EXTRACELLULAR PROTEINS

Cystine depleted cystinotic fibroblasts reaccumulate cystine when incubated in cystine free medium supplemented with a variety of disulfide (cystine)-containing proteins. The cystine accumulation is linear with the concentration of added protein. Correlation of the amount of degradation of 125I labelled protein with cystine accumulation yields a significant correlation (r=0.97 for bovine serum albumin [BSA], and r=0.81 for bovine insulin). Similar results have been found for human and rat albumin, and bovine y globulin & thyroglobulin. This cystine accumulation is inhibited by inhibitors of proteolysis, and inhibitors of selective endocytosis. Reduced BSA (RBSA) produced by pre-treatment with mercaptoethanol in 6 M urea yields diminished cystine accumulation even though more of the reduced protein is degraded:0.18 nmol/106 cells cystine accumulated and 0.50 mg/106 cells RBSA degraded in 24 hrs; 0.70 nmol/106 cells cystine accumulated and 0.20 mg BSA degraded in 24 hrs. Ovalbumin (OVA), which contains 4 mol of cysteine/mol of protein and 1 mol cystine yields no cystine accumulation after 24 hrs incubation in which 0.86 mg/106 cells OVA was degraded. Addition of OVA or RBSA to non-depleted cystinotic fibroblasts produces cystine depletion (30% in 4 hrs, 55% in 24 hrs). We conclude that cystinotic cells accumulate cystine through the lysosomal retention of cystine, but not cysteine residues following their release from peptide chains during proteolysis. Supported by USPHS AM 25548.

Xenoantibody directed against molecular components of the HLA system. Quantitation of antibody to serologically defined HLA complexes on the cell surface.

A quantitative absorption-blocking assay was used to measure the levels of antibody in immune rabbit sera that react with the serologically defined HLA molecular complex on the cell surface. Quantitation was determined by comparing the number of xenoantibody-treated cells with the number of untreated control cells needed to absorb a defined amount of cytolytic alloantibody. Complement-dependent cytolytic titers were used to standardize the total antibody content reactive with cell surface antigens of xenoantisera used to treat absorbing cells. The ability of xenoantibody to "block" or inhibit the reaction of alloantibody with the absorbing cell was observed to be the result of concomitant removal of both xeno- and alloantigenic determinants from the cell surface. Immunofluorescence data with xenoantibody suggested that removal was accomplished by temperature-dependent selective redistribution and endocytosis of the serologically defined HLA molecule complex. By using these procedures, xenoantisera have been characterized that contain different amounts of antibody to the two-component HLA complex relative to their total cytolytic antibody content. Variation ranges from a xenoantiserum with little or no blocking capacity to several highly effective serologically defined HLA blocking xenoantisera.