Processing Of N-linked Glycoproteins Research Articles

The Antipsychotic Drug Penfluridol Inhibits N-Linked Glycoprotein Processing and Enhances T-cell-Mediated Tumor Immunity.

Aberrant N-linked glycosylation is a prominent feature of cancers. Perturbance of oligosaccharide structure on cell surfaces directly affects key processes in tumor development and progression. In spite of the critical role played by N-linked glycans in tumor biology, the discovery of small molecules that specifically disturbs the N-linked glycans is still under investigation. To identify more saccharide-structure-perturbing compounds, a repurposed drug screen by using a library consisting of 1530 FDA-approved drugs was performed. Interestingly, an antipsychotic drug, penfluridol, was identified as being able to decrease cell surface wheat germ agglutinin staining. In the presence of penfluridol, cell membrane glycoproteins programmed death-ligand 1 (PD-L1) shifted to a lower molecular weight. Further studies demonstrated that penfluridol treatment caused an accumulation of high-mannose oligosaccharides, especially Man5-7GlcNAc2 glycan structures. Mechanistically, this effect is due to direct targeting of MAN1A1 mannosidase, a Golgi enzyme involved in N-glycan maturation. Moreover, we found that altered glycosylation of PD-L1 caused by penfluridol disrupted interactions between programmed cell death protein 1 and PD-L1, resulting in activation of T-cell tumor immunity. In a mouse xenograft and glioma model, penfluridol enhanced the antitumor effect of the anti-PD-L1 antibody in vivo. Overall, these findings revealed an important biological activity of the antipsychotic drug penfluridol as an inhibitor of glycan processing and proposed a repurposed use of penfluridol in antitumor therapy through activation of T-cell immunity.

P32 regulates ER stress and lipid homeostasis by down-regulating GCS1 expression.

Sustained endoplasmic reticulum (ER) stress plays a major role in the development of many metabolic diseases, including cardiovascular disease, nonalcoholic fatty liver disease, insulin resistance, obesity, and diabetes. p32 is a multicompartmental protein involved in the regulation of oxidative phosphorylation and glucose oxidation. p32 ablation is associated with resistance to age-associated and diet-induced obesity through a mechanism that remains largely unknown. Here, we show that p32 promotes lipid biosynthesis by modulating fatty acid-induced ER stress. We found that p32 interacts with endoplasmic reticulum-anchored enzyme mannosyl-oligosaccharide glucosidase I (GCS1), an ER lumen-anchored glucosidase that is essential for the processing of N-linked glycoproteins, and reduces GCS1 in a lysosome-dependent manner. We demonstrate that increased GCS1 expression alleviates fatty acid-induced ER stress and is critical for suppressing ER stress-associated lipogenic gene activation, as demonstrated by the down-regulation of Srebp1, Fasn, and Acc. Consistently, suppression of p32 leads to increased GCS1 expression and alleviates fatty acid-induced ER stress, resulting in reduced lipid accumulation. Thus, p32 and GCS1 are regulators of ER function and lipid homeostasis and are potential therapeutic targets for the treatment of obesity and diabetes.-Liu, Y., Leslie, P. L., Jin, A., Itahana, K., Graves, L. M., Zhang, Y. p32 regulates ER stress and lipid homeostasis by down-regulating GCS1 expression.

Glycoside Hydrolase MoGls2 Controls Asexual/Sexual Development, Cell Wall Integrity and Infectious Growth in the Rice Blast Fungus.

N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs) are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis. Target deletion of MoGLS2 in M. oryzae resulted in a reduced mycelial growth, an increased conidial production, delayed conidial germination and loss the ability of sexual reproduction. Pathogenicity assays revealed that the ΔMogls2 mutant showed significantly decreased in virulence and infectious growth. Further studies showed that the mutant was less sensitive to salt and osmotic stress, and increased sensitivity to cell wall stresses. Additionally, the ΔMogls2 mutant showed a defect in cell wall integrity. Our results indicate that MoGls2 is a key protein for the growth and development of M. oryzae, involving in the regulation of asexual/sexual development, stress response, cell wall integrity and infectious growth.

ChemInform Abstract: Synthesis and Biological Activity of Naturally Occurring α‐Glucosidase Inhibitors

AbstractReview: 176 refs.

Alpha-1,2-Mannosidase and Hence N-Glycosylation Are Required for Regulatory T Cell Migration and Allograft Tolerance in Mice

BackgroundSpecific immunological unresponsiveness to alloantigens can be induced in vivo by treating mice with a donor alloantigen in combination with a non-depleting anti-CD4 antibody. This tolerance induction protocol enriches for alloantigen reactive regulatory T cells (Treg). We previously demonstrated that alpha-1,2-mannosidase, an enzyme involved in the synthesis and processing of N-linked glycoproteins, is highly expressed in tolerant mice, in both graft infiltrating leukocytes and peripheral blood lymphocytes.Principal FindingsIn this study we have identified that alpha-1,2-mannosidase expression increases in CD25+CD4+ Treg when they encounter alloantigen in vivo. When alpha-1,2-mannosidase enzyme activity was blocked, Treg retained their capacity to suppress T cell proliferation in vitro but were unable to bind to physiologically relevant ligands in vitro. Further in vivo analysis demonstrated that blocking alpha-1,2-mannosidase in Treg resulted in the migration of significantly lower numbers to the peripheral lymph nodes in skin grafted mice following adoptive transfer, where they were less able to inhibit the proliferation of naïve T cells responding to donor alloantigen and hence unable prevent allograft rejection in vivo.SignificanceTaken together, our results suggest that activation of alloantigen reactive Treg results in increased alpha-1,2-mannosidase expression and altered N-glycosylation of cell surface proteins. In our experimental system, altered N-glycosylation is not essential for intrinsic Treg suppressive capacity, but is essential in vivo as it facilitates Treg migration to sites where they can regulate immune priming. Migration of Treg is central to their role in regulating in vivo immune responses and may require specific changes in N-glycosylation upon antigen encounter.

N-acetylglucosaminyltransferase V Modifies TrKA Protein, Regulates the Receptor Function

1. N-Acetylglucosaminyltransferases V (GnT-V/Mgat5) play a pivotal role in the processing of N-linked glycoproteins in the Golgi apparatus. The aim of the present study is to investigate whether the N-acetylglucosaminyltransferase V is able to modify TrKA, the high-affinity tyrosine kinase-type receptor for NGF, and thereby to regulate the receptor function. 2. Plasmids of the pcDNA3/GnT-V and pcDNA3 were transfected into PC12 cells. Expression of GnT-V protein was detected by Western blot. TrKA protein was examined by immunoprecipitation. Endocytosis of TrKA was investigated by the method of receptor internalization. 3. We report here that over-expression GnT-V directly modifies TrKA protein, accompanied by marked enhancement of axon outgrowth in rat pheochromocytoma cells (PC12) elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. Further study indicated that modification of TrKA glycoprotein could directly enhance NGF-activated autophosphorylation of immunoprecipitated TrKA in vitro. To further elucidate the mechanism, we study the different time point of endocytosis of TrKA receptor. The results show that TrKA of GnT-V gene-transfected PC12 Cells delayed their removal by constitutive endocytosis as compared to the mock cells, suggesting high expression of GnT-V may affect their receptor TrKA endocytosis. 4. These results strongly suggest that N-acetylglucosaminyltransferase V functioning as a specific endogenous role of NGF receptor function, which appear to be due, at least in part, to the promotion of differentiation. This work is an important step toward intriguing innovative therapeutic strategies targeting glycosyltransferase.

Deficiency of ATP2C1, a Golgi Ion Pump, Induces Secretory Pathway Defects in Endoplasmic Reticulum (ER)-associated Degradation and Sensitivity to ER Stress

Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarily affects keratinocytes. ATP2C1-encoded protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting in defects in both post-translational processing of wild-type thyroglobulin (a secretory glycoprotein) as well as endoplasmic reticulum-associated protein degradation of mutant thyroglobulin, whereas degradation of a nonglycosylated misfolded secretory protein substrate appears unaffected. Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone proteins, nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ER stress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. These data point to the important contributions of the Golgi-localized ATP2C1 protein in homeostatic maintenance throughout the secretory pathway.

Human cytokine production against cattle

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal α-1,2–linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(α1-2)Glc(α1-3)Glc(α1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G→C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T→C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G→C transition, whereas the father was heterozygous for the T→C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the α-glucosidase I deficiency and the disease is postulated.

A Novel Disorder Caused by Defective Biosynthesis of N-Linked Oligosaccharides Due to Glucosidase I Deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.

Mice Lacking Dad1, the Defender against Apoptotic Death-1, Express Abnormal N-Linked Glycoproteins and Undergo Increased Embryonic Apoptosis

Dad1 has been shown to play a role in preventing apoptotic cell death and in regulating levels of N-linked glycosylation in Saccharomyces cerevisiae and the BHK hamster cell line. To address the in vivo role of Dad1 in these processes during multicellular development, we have analyzed mice carrying a null allele for Dad1. Embryos homozygous for this mutation express abnormal N-glycosylated proteins and are developmentally delayed by embryonic day 7.5. Such mutants exhibit aberrant morphology, impaired mesodermal development, and increased levels of apoptosis in specific tissues. These defects culminate in homozygous embryos failing to turn the posterior axis and subsequent lethality by embryonic day 10.5. Thus, Dad1 is required for proper processing of N-linked glycoproteins and for certain cell survival in the mouse.

Glycosyltransferase Genes: Applications to Medical Science.

Glycosylation represents a major posttranslational modification reaction of proteins and plays a pivotal role in a variety of cellular events. N-Acetylglucosaminyltransferases III (GnT-III) and V (GnT-V), and α 1-6 fucosyltransferase are key enzymes in the processing of N-linked glycoproteins, and are heavily involved in cancer progression and metastasis. Very recently we also found that GnT-III is capable of reducing or masking the expression of the αGal epitope, which is a major xenoantigen in swine to human xenotransplantation. In this minireview we focus on the role of the above glycosyltransferases in cancer metastasis as well as in xenotransplantation.

Synthesis of cell-wall glycoproteins and their characterization in oat coleoptiles

d-[U- 14C]Glucosamine was rapidly taken up by oat coleoptile segments and metabolized to radioactive UDP- N-acetylglucosamine, which acted as specific glycosyl donor for the synthesis of glycolipids and cytosolic, membrane-bound and cell-wall glycoproteins. Cell-wall glycoproteins were solubilized from the walls by either cell-wall-degrading enzymes or chemical extractants. The solubilized cell-wall glycoproteins in the presence of peptide N-glycosidase F released oligosaccharide chains higher than seven glycosidic residues. The combined action of peptide N-glycosidase F and N-acetyl-β- d-glucosaminidase on cell-wall glycoproteins indicated the presence of N-acetylglucosamine residues β-1,2-linked to mannose. Less than 9% of the radioactive oligosaccharide chains was released from the solubilized cell-wall glycoproteins when treated with 0.5 M NaOH at 20°, whereas more than 45% of the radioactivity was released in the presence of 1 M NaOH at 50°. The high hydrolytic sensitivity of cell-wall glycoproteins to peptide N-glycosidase F, N-acetyl-β- d-glucosaminidase NaOH at 50° indicated that most N-acetylglucosamine residues were incorporated into N-linked cell-wall glycoproteins. Further evidence of this was obtained by the use of inhibitors of biosynthesis and processing of N-linked glycoproteins.

Synthesis of oligosaccharide substrates for N-linked glycoprotein processing enzymes

The stereoselective syntheses of one pentasaccharide and one tetrasaccharide containing the Glc- α-(1 → 3)-Man- α moiety as their terminal unit, as well as one tetrasaccharide and one trisaccharide containing the Man- α-(1 → 2)-Man- α terminal unit were accomplished through the utilization of two key glycosyl donors, namely, 4-pentenyl 3-O- acetyl-2,4,6- tri-O- benzyl-α- d- mannopyranoside and ethyl 2-O- acetyl-3,4,6- tri-O- benzyl-1-thio-α- d-mannopyranoside .

Disaccharides as endomannosidase inhibitors: syntheses of alpha-homomannojirimycin and beta-homomannojirimycin linked to D-glucose and D-mannose.

4-O-(alpha-D-Glucopyranosyl)-alphaHMJ (Glcalpha1,4HMJ), 4-O-(alpha-mannopyranosyl)-alphaHMJ (Manalpha1,4alphaHMJ), 4-0-(alpha-glucopyranosyl)-betaHMJ (Glcalpha1,4betaHMJ), and 4-0-(alpha-mannopyranosyl)-betaHMJ (Manalpha1, 4betaHMJ) were synthesized as endomannosidase inhibitors which are potentially useful both for probing the pathways of N-linked glycoprotein processing and for the chemotherapy of some viral diseases.

Glycoprotein synthesis in yeast. Early events in N-linked oligosaccharide processing in Schizosaccharomyces pombe.

Oligosaccharide-lipid precursors and glycoprotein N-linked oligosaccharides isolated from the fission yeast, Schizosaccharomyces pombe, were compared with those from the budding yeast, Saccharomyces cerevisiae. Bio-Gel P-4 chromatography of oligosaccharide intermediates showed that Glc3Man9GlcNAc2-PP-dol synthesis, transfer of glycan to protein, and glucose removal to yield Man9GlcNAc2 proceeded in S. pombe as in S. cerevisiae. Two series of oligosaccharides were released from S. pombe glycoproteins by endo-beta-N-acetylglucosaminidase H; large "mannan-like" structures and smaller precursor or "core-filling" species. Unexpectedly, the smallest S. pombe N-linked glycan was Man9GlcNAc, confirmed by 500 MHz 1H NMR spectroscopy to be the lipid-linked isomer. No endoplasmic reticulum Man9-alpha 1,2-mannosidase activity was detected in S. pombe, thus identifying Man9GlcNAc as the minimum precursor for oligosaccharide elongation in contrast to the Man8GlcNAc2 intermediate identified in S. cerevisiae (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). S. pombe Hex10GlcNAc was at least four isomers by high pH anion-exchange chromatography with pulsed amperometric detection. Compositional analyses identified two of the major species as GalMan9GlcNAc and GlcMan9GlcNAc, the latter of which suggests that glycan trimming may be attenuated in the S. pombe endoplasmic reticulum. Hex13GlcNAc from S. pombe was homogeneous by mass spectrometry but yielded 12 species by high pH anion-exchange chromatography. Compositional analyses, alpha-galactosidase digestion, and lectin affinity chromatography on Griffonia simplicifolia lectin I-agarose indicated these to be a family of GalxMan13-xGlcNAc isomers (X = 1-4 residues). The absence of Man9GlcNAc2 to Man8GlcNAc2 trimming in S. pombe and elongation of the lipid precursor of Man9GlcNAc with both Man and Gal to form "galactomannans" provides a novel system for N-linked glycoprotein processing studies.

Synthesis of 1,5-dideoxy-3-O-(α-D-mannopyranosyl)-1,5-imino-D-mannitol and 1,5-dideoxy-3-O-(α-D-glucopyranosyl)-1,5-imino-D-mannitol: Powerful inhibitors of endomannosidase

The synthesis and conformations of 3-O-methylDMJ, 3-O-(α-D-glucopyranosyl)-DMJ (Glcα1,3DMJ), and 3-O-(α-D-mannopyranosyl)-DMJ (Manα1,3DMJ) are described. Manα1,3DMJ and Glcα1,3DMJ are shown to be very powerful inhibitors of an endomannosidase. The potential use of these compounds in both probing the pathways of N-linked glycoprotein processing and in the chemotherapy of some viral diseases is discussed.

Glycoprotein processing is required for completion but not initiation of oligodendroglial differentiation from its bipotential progenitor cell.

The role of N-linked glycoproteins in the development of oligodendroglia has been studied in a culture system that initially contains the progenitor cell for oligodendroglia and type 2 astrocytes. The progenitor cells, derived from mixed glial primary cultures of newborn rat cerebrum, were studied under culture conditions that we have shown previously to induce oligodendroglial differentiation. Castanospermine was used to inhibit processing of N-linked glycoproteins by its inhibitory action on glucosidase I, the enzyme responsible for the initial trimming of glucose residues from the glucosylated high mannose core oligosaccharide derived from the dolichol pathway. Exposure to castanospermine had no effect on the initial commitment of the progenitors to oligodendroglial differentiation, i.e. 95% of both control and castanospermine-treated cells became galactocerebroside (GC) positive. However, the developmental inductions of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH) and the elaboration of a network of fine interconnecting processes were prevented by the castanospermine exposure. No effect of castanospermine on cell number was observed. A major effect of the inhibitor on glycoprotein processing was manifested by an accumulation of high mannose glycoproteins, of abnormal oligosaccharide structure, compatible with the inhibition of glucosidase I.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibition of platelet aggregation of metastatic H- ras-transformed [formula omitted] fibroblasts with castanospermine, an N-linked glycoprotein processing inhibitor

A series of T24-H-ras-transformed 10T 1 2 fibroblasts with varying metastatic potential was tested for the ability to aggregate platelets. Results indicate that although platelet activation was always detected in the highly metastatic cells, some non-metastatic cells also have the ability to cause platelet aggregation, suggesting that this is a necessary but not sufficient characteristic of the metastatic phenotype. Apyrase, an ADP scavenger, effectively inhibited platelet aggregation by metastatic cells, however, there was no significant increase in ADP secretion or relation to the ability of the tumor cells to activate platelets. Hirudin, a thrombin inhibitor, did not affect aggregation, suggesting that the pathway of activation is thrombin-independent. The glycoprotein processing inhibitor, castanospermine, which reduces glycosidase I activity and metastatic capability, inhibited the ability of metastatic cells to cause platelet aggregation. However, another inhibitor of oligosaccharide processing, swainsonine, which inhibits mannosidase II activity and does not reduce metastasis, had no effect on platelet aggregation. These results show that the integrity of N-linked oligosaccharide structure of glycoproteins is an important feature of the ability of ras-transformed fibroblasts to activate platelets.

Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain.

We have analyzed the functional domain structure of rat mammary glucosidase I, an enzyme involved in N-linked glycoprotein processing, using biochemical and immunological approaches. The enzyme contains a high mannose type sugar chain that can be cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity. Based on trypsin digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain. A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes with trypsin. This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific affinity gel. Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme, consistent with its location as an integral membrane protein, whereas the 39-kDa fragment partitioned in the aqueous phase, a characteristic of soluble polypeptide. These results indicate that glucosidase I is a transmembrane protein with a luminally oriented catalytic domain. Such an orientation of the catalytic domain may facilitate the sequential processing of asparagine-linked oligosaccharide, soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum.

Developmental regulation of glucosidase I, an enzyme involved in the processing of asparagine-linked glycoproteins in rat mammary gland.

Glucosidase I involved in the processing of N-linked glycoproteins was purified to homogeneity from the lactating rat mammary gland. The purified enzyme exhibited a single band at 85 kDa on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Polyclonal antibodies raised against the enzyme recognized a similar band on Western blots and also inhibited the enzyme activity. The enzyme levels gradually increased until the midlactation stage and thereafter declined sharply during the period of postlactation. A similar profile of the levels of immunoreactive glucosidase I was observed. These findings suggest that the accumulation of glucosidase I is modulated as a function of gland ontogeny. The results on hormonal regulation of glucosidase I indicate that the synthesis of the enzyme is stimulated by a combination of insulin, hydrocortisone, and prolactin; additionally, epidermal growth factor may play a role in this regulation. The above observation was substantiated by immunoprecipitation of [35S]methionine-labeled microsomal extracts with anti-glucosidase I antibodies. The immunoprecipitation of soluble extracts from [35S]methionine-labeled tissue with anti-rat alpha-lactalbumin antibodies indicates that these hormones not only stimulate the synthesis of alpha-lactalbumin but also play an important role in its glycosylation.