Nonsyndromic Mental Retardation Research Articles

Autozygosity mapping in consanguineous Pakistani families identifies nine non-overlapping novel linkage intervals for autosomal recessive non-syndromic mental retardation (AR-NSMR); shows genetic heterogeneity for AR-NSMR.

Psychological disturbance (PD) or cerebral dysfunction (CD) covers several clinical areas, and has defining features of mental retardation. Recently, we conducted a study to investigate heritable heterogeneity in Pakistani consanguineous couples with recessive autosomal intellectual abnormalities. A cohort of three consanguineous families with multiple birth defects, belonging two to district lower Dir and one to district Lodhra, were selected for molecular analysis. All the affected individuals in the cohort showed autosomal recessive non-syndromic mental disturbances. DNA was extracted and subjected to Single tagged sequence (STS) marker analyses to all known non-syndromic autosomal recessive mental retardation (NS-ARMR) genes, while autozygosity mapping was performed by advanced SNP techniques. Fragment analyses of the NS-ARMR disease genes CRBN, CC2D2A, PRSS12, GRIK2, TUSC3, and CC2D1A using polymorphic STS markers confirmed these to be contender genes for the alteration. Mapping of autozygosity in all the study subjects using genome study revealed nine novel linkage intervals, i.e. four intervals for MR4, two intervals for MR8 and three intervals for MR13. In spite of being a monogenic condition, autosomal recessive mental retardation shows genetic heterogeneity and several genes are involved in different families; hence, there is a chance for involvement of separate gene in each family.

Transcriptomic profiling of mice brain under Bex3 regulation.

BEX family genes are expressed in various tissues and play significant roles in neuronal development. A mouse model of Bex3 gene knock-out was generated in this study, using the CRISPR-Cas9 system. Transcriptomic analysis of the brain was performed to identify genes and pathways under Bex3 regulation. Essential biological functions under the control of Bex3 related to brain development were identified. Ninety-five genes were differentially expressed under Bex3-/- regulation, with 53 down and 42 up. Among down-regulated genes, LOC102633156 is a member of zf-C2H2, Xlr3a is an X-linked lymphocyte regulated gene, LOC101056144 is a hippocampal related gene, 2210418O10Rik and Fam205a3 are cortex related genes. Among the upregulated genes, Zfp967 is a zf protein, Tgtp2 is a T cell-specific regulator, Trpc2 is a neuron-related gene, and Evi2 is related to NF1. A total of 34 KEGG disease terms were identified under the Bex3-/- regulation. The most prominent is non-syndromic X-linked mental retardation, where Fgd1 is enriched. Similarly, IRF, MBD, SAND, zf-BED, and zf-C2H2 were significantly enriched transcription factors. A further study is required to confirm and explain each aspect that has been identified in this study.

Синдром протяженной делеции при тяжелой форме мукополисахаридоза II типа

В статье отражены современные представления о сочетании нескольких наследственных синдромов в результате протяженной делеции Х хромосомы на примере мукополисахаридоза II типа и умственной отсталости. Проанализированы описания встречающихся в литературе сочетаний моногенных синдромов, обозначаемых как «сопряженные» или «протяженные» генные синдромы, а также понятие «смежные» гены. Протяженные генные синдромы, обусловленные делецией участка X хромосомы у мальчиков, приводят к структурной и функциональной нуллисомии, поэтому необходимы исследования числа копий генов с целью определения точного генетического дефекта. Суммированы данные литературы и собственные наблюдения, касающиеся вопросов этиологии, патогенеза, сроков манифестации и клинической симптоматики болезни, методов диагностики, современного лечения и профилактики мукополисахаридоза II типа. Мукополисахаридоз II типа относится к заболеваниям из группы лизосомных болезней накопления с мультиорганным поражением. Описан клинический случай синдрома Хантера с протяженной делецией хромосомы Х и ранним дебютом клинических проявлений комбинированного стеноза позвоночного канала и миелопатии шейного отдела спинного мозга вследствие накопления гликозаминогликанов (ГАГ) в оболочках спинного мозга. Смешанный тетрапарез при мукополисахаридозе II типа наблюдается реже, чем при других мукополисахаридозах и, как правило, манифестирует в более старшем возрасте. Высказано предположение, что тяжелый фенотип с сочетанием неврологической симптоматики и умственнной отсталости связан с протяженной делецией хромосомы Х, включающей полную делецию гена IDS. В алгоритм обследования пациентов с несиндромальной умственной отсталостью входят анализ кариотипа, ДНК-диагностика синдрома ломкой хромосомы Х и хромосомный микроматричный анализ. Показано, что при обследовании мальчиков с умственной отсталостью в этот алгоритм дополнительно следует включить биохимические тесты - определение ГАГ в моче и анализ ферментов лизосом, что позволит с большей эффективностью выявлять больных с мукополисахаридоз II типа. This article presented below reflects modern ideas about the combination of several hereditary syndromes as a result of an contiguous deletion of the X chromosome by the example of mucopolysaccharidosis type II and mental retardation. Monogenic syndromes occurring in the literature in combination, marked as contiguous gene syndromes, have been analyzed, along with what the notion of contiguous geneshas been described. Contiguous gene syndromes due to the deletion of the X chromosome region in boys lead to structural and functional nullisomy, thus confirming the significance of studying the number of copies of genes in order to determine the exact genetic defect. The data of the literature and our own observations concerning issues of etiology, pathogenesis, time of manifestation and clinical symptoms of the disease, diagnostic methods, treatment and prevention of mucopolysaccharidosis type II are summarized. Mucopolysaccharidosis type II refers to а group of lysosomal storage diseases with multiorgan lesions. Here is described a clinical case of Hunter syndrome with a long deletion of the X chromosome and an early manifestation of a combined stenosis of the spinal canal, the development of myelopathy of the cervical spinal cord as a result of the accumulation of glycosaminoglycans in the membranes of the spinal cord. The described case with the clinical picture of mixed tetraparesis is a rarer symptom with type II mucopolysaccharidosis than with other mucopolysaccharidosis and, as a rule, manifests at an older age. It was suggested that such a phenotypic correlation in patients with mucopolysaccharidosis type II, as a severe phenotype with neurological symptoms with a complete deletion of the IDS gene. The algorithm for examining patients with non-syndromic mental retardation includes the analysis of karyotype, DNA-diagnosis of fragile X chromosome, and chromosome analysis. It is shown that a survey is needed to exclude MPS II in boys with mental retardation and the inclusion of simple biochemical tests in these algorithms - determination of GAG in urine and analysis of lysosomal enzymes, which will more effectively identify patients with MPS type II.

VARIETY OF CLINICAL MANIFESTATIONS IN MUTATIONS IN THE DYNC1H1 GENE

Introduction. To date, DYNC1H1 gene mutations are known for large number of hereditary diseases. It is believed that different mutations have variable effects to protein function and, accordingly, to various clinical manifestations. Results. There are a clinical and genetic characteristics of two Russian patients with two types of diseases: spinal muscular atrophy with predominant lesion of the lower extremities (SMALED) and non-syndromic mental retardation type 13 (MR13) in combination with a brain malformations and epilepsy due to newly identified mutations in the DYNC1H1 gene. Conclusion There is some evidence in support of the hypothesis that the amino acid sequence changing in the tail domain of dynein lead to the appearance of SMALED, and in the motor domain lead to MR13. Exome or genome sequencing are required as the main method for their diagnosis due to the high genetic heterogeneity of non-syndromic MR and SMALED, the lack of specific clinical markers and hotspot mutations in the DYNC1H1 gene.

Cereblon Control of Zebrafish Brain Size by Regulation of Neural Stem Cell Proliferation

SummaryThalidomide is a teratogen that causes multiple malformations in the developing baby through its interaction with cereblon (CRBN), a substrate receptor subunit of the CRL4 E3 ubiquitin ligase complex. CRBN was originally reported as a gene associated with autosomal recessive non-syndromic mild mental retardation. However, the function of CRBN during brain development remains largely unknown. Here we demonstrate that CRBN promotes brain development by facilitating the proliferation of neural stem cells (NSCs). Knockdown of CRBN in zebrafish embryos impaired brain development and led to small brains, as did treatment with thalidomide. By contrast, overexpression of CRBN resulted in enlarged brains, leading to the expansion of NSC regions and increased cell proliferation in the early brain field and an expanded expression of brain region-specific genes and neural and glial marker genes. These results demonstrate that CRBN functions in the determination of brain size by regulating the proliferation of NSCs during development.

Diversity and Prevalence of Hereditary Diseases among Nogais of the Karachay-Cherkess Republic

The diversity and prevalence of hereditary diseases (HDs) among Nogais of the Karachay-Cherkess Republic (KChR) are described. The size of the surveyed KChR population was 387231 individuals, including 3.81% Nogais (14741 individuals). We revealed 36 nosological forms of HDs (110 patients from 81 families): 22 with autosomal dominant (AD) inheritance, 10 with autosomal recessive (AR) inheritance, and 4 with X-linked inheritance. The prevalence of HDs in Nogais was 1: 134. The features of HD diversity in Nogais were determined in comparison with the previously surveyed populations of Russia. The accumulation of Ehlers–Danlos syndrome (1: 388), AD amelogenesis imperfecta (1: 3685), AD ichthyosis (1: 4914), AR nonsyndromic mental retardation (1: 1340), AR Gilbert syndrome (1: 4914), and X-linked inherited deficit of glucose-6-dehydrogenase (1: 1774 males) was established. The analysis of heterozygous carriage of mutations “major” for Russia in the genes of the four following AR diseases in 118 unrelated clinically healthy Nogais (236 analyzed chromosomes) was performed: cystic fibrosis (13 mutations in the CFTR gene: CFTRdele2,3 (21 kb), F508del, I507del, 1677delTA, 2184insA, 2143delT, 2183AA>G, 2184delA, 394delTT, 3821delT, L138ins, E92K, W1282X); phenylketonuria (six frequent mutations in the PAH gene: R261X, R408W, R413P, F331S, P211T, P211L); nonsyndromic sensorineural hearing loss (35delG mutation in the GJB2 gene); and Gilbert syndrome (an increase in the number of TA repeats in the UGT1A1 gene). Allelic specificity for all studied genes in the Nogai people was revealed.

New allelic variants of non-syndromic mental retardation of type 20 caused by mutations in the MEF2C gene

To determine clinical and genetic characteristics of patients with non-syndromic mental retardation (NMR), type 20 with autosomal dominant type of inheritance (OMIM: 613443). Fourteen patients were studied including four patients with mutations in the MEF2C gene revealed by exome sequencing. Three of the four mutations in the gene were found for the first time. Based on a comparative analysis of the clinical manifestations of 4 observed patients and 9 patients with type 20 NMR described in the literature, the authors determined common clinical characteristics of this syndrome. In most cases with delayed psycho-speech development and convulsive syndrome, the patients were expected to have various variants of early epileptic encephalopathies, in which presence of convulsive paroxysms leads to intellectual deficit, while in the case of NMR, the delay in development can be noted long before the onset of seizures. Exome sequencing is the most effective method of NMR diagnosis.

Cereblon Control of Zebrafish Brain Size by Regulation of Neural Stem Cell Proliferation

Thalidomide is a teratogen that causes multiple malformations when exposed to developing vertebrate embryos. This was recently shown to be due to its interaction with Cereblon (CRBN), a novel substrate receptor subunit of the CRL4 E3 ubiquitin ligase complex, whose enzymatic activity is controlled by thalidomide and its analogs. CRBN was originally reported as a gene associated with autosomal recessive non-syndromic mild mental retardation. However, the function of CRBN during brain development remains largely unknown. Here we demonstrate that CRBN promotes brain development by facilitating proliferation of neural stem cells (NSCs). Knockdown of CRBN in zebrafish embryos impaired brain development and led to small brains, as did treatment with thalidomide. By contrast, overexpression of CRBN resulted in enlarged brains, leading to the expansion of NSC regions in the telencephalon, the diencephalon and the midbrain, increased cell proliferation throughout the brain, and an expanded expression of brain region-specific genes and neural and glial marker genes. CRBN functions downstream of Six3 and Lhx2 to regulate neural stem cell proliferation. These results demonstrate a new function for CRBN in the determination of brain size by regulating proliferation of NSCs during development.

Cereblon: A Protein Crucial to the Multiple Functions of Immunomodulatory Drugs as well as Cell Metabolism and Disease Generation.

It is well known that cereblon is a key protein in autosomal recessive nonsyndromic mental retardation. Studies have reported that it has an intermediary role in helping immunomodulatory drugs perform their immunomodulatory and tumoricidal effects. In addition, cereblon also regulates the expression, assembly, and activities of other special proteins related to cell proliferation and metabolism, resulting in the occurrence and development of metabolic diseases. This review details the multiple functions of cereblon and the underlying mechanisms. We also put forward some unsolved problems, including the intrinsic mechanism of cereblon function and the possible regulatory mechanisms of its expression.

Cereblon in health and disease

Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that has been linked to autosomal recessive non-syndromic mental retardation. Several key findings suggest diverse roles of CRBN, including its regulation of the large-conductance calcium- and voltage-activated potassium (BKCa) channels, regulation of thalidomide-binding proteins, and mediation of lenalidomide treatment in multiple myeloma. Recent studies also indicate that CRBN is involved in energy metabolism and negatively regulates AMP-activated protein kinase signaling. Mice with genetic depletion of CRBN are resistant to various stress conditions including a high-fat diet, endoplasmic reticulum stress, ischemia/reperfusion injury, and alcohol-related liver damage. In this review, we discuss the various roles of CRBN in human health and disease and suggest avenues for further research to enhance our basic knowledge and clinical application of CRBN.

TUSC3 promotes colorectal cancer progression and epithelial-mesenchymal transition (EMT) through WNT/β-catenin and MAPK signalling.

Colorectal cancer (CRC) is one of the most common malignancies and is the second leading cause of cancer death in humans. Tumour suppressor candidate 3 (TUSC3) plays an important role in embryogenesis and metabolism. Deletion of TUSC3 often causes non-syndromic mental retardation. Even though TUSC3 deregulation is frequently observed in epithelial cancers, the function of TUSC3 in CRC has remained unknown. In this study, we observed greater expression of TUSC3 at the mRNA and protein level in clinical colorectal tumour samples compared with paired normal tissues. Gain- and loss-of-function analyses were performed to evaluate the functional significance of TUSC3 in CRC initiation and progression. Immunoblotting, immunofluorescence, and co-immunoprecipitation analyses were used to identify potential pathways with which TUSC3 might be involved. Overexpression of TUSC3 in CRC cells induced epithelial-mesenchymal transition (EMT) in CRC cells, accompanied by down-regulation of the epithelial marker, E-cadherin, and up-regulation of the mesenchymal marker, vimentin. Increased proliferation, migration, and invasion, as well as accelerated xenograft tumour growth, were observed in TUSC3-overexpressing CRC cells, while opposite effects were achieved in TUSC3-silenced cells. In conclusion, our study demonstrated the oncogenic role of TUSC3 in CRC and showed that TUSC3 may be responsible for alternations in the proliferation ability, aggressiveness, and invasive/metastatic potential of CRC through regulating the MAPK, PI3K/Akt, and Wnt/β-catenin signalling pathways.

Neto2 Influences on Kainate Receptor Pharmacology and Function.

Neuropilin tolloid-like protein 2 (Neto2) is an auxiliary subunit of kainate receptors (KARs). It specifically regulates KARs, for example slows desensitization and deactivation, increases the rate of recovery from desensitization, promotes modal gating and increases agonist sensitivity. Although the mechanism of Neto2 modulation is still unclear, gain-of-function results from the characterization of GluK1-GluA2 chimeras indicate that the GluK1 sequences included in these chimeras (part or all of the TMD and part of the linkers between the TMDs and LBD) play a key role in Neto2 modulation of KAR. In addition, GluK2 M3-S2 linkers and the D1-D1 dimer interface were also recently identified to be important for Neto2 modulation, and some studies suggested that Neto2's N-terminal regions, LDLa domain and the C-terminal regions are important for its modulation of KARs. Although more studies are needed to confirm the roles of these domains and to identify all the domains and residues essential for KAR modulation, these results facilitate our understanding of Neto2 modulation at the structural level, which could potentially aid the development of novel therapies for the treatment of diseases that are associated with KARs, for example epilepsies, non-syndromic autosomal recessive mental retardation, schizophrenia and bipolar disorder.

Computational Analysis and Polymorphism study of Tumor Suppressor Candidate Gene-3 for Non Syndromic Autosomal Recessive Mental Retardation

Computational Analysis and Polymorphism study of Tumor Suppressor Candidate Gene-3 for Non Syndromic Autosomal Recessive Mental Retardation Mental Retardation (MR) is regarded as a neuronal malfunction characterized by a low Intellectual Quotient (IQ). To date, few genes (GRIK2, TUSC3, TRAPPC9, TECR, ST3GAL3, MED23, MAN1B1, NSUN1 PRSS12, CRBN, CC2D1A) for autosomal-recessive non syndromic MR (NS-ARMR) have been identified and established in various families with MR. The recently reported candidate gene “Tumor Suppressor Candidate Gene-3 (TUSC3)” was selected for computational analysis to explore its potential role in pathology as it is the only gene for MR reported in more than five different familial cases worldwide. TUSC3 gene located at chromosome 8p22 contains 11 exons and encodes a protein involved in the vertebrate plasma membrane magnesium ion transport system. Three dimensional structures of the candidate gene TUSC3 and its mutated structure with Q55X were generated. Template parameters were based on Z-score, E value, resolution and quality. Present study demonstrates the reliability of 3D model which satisfies the overall quality of the model constructed, employing both physiochemical and statistical model evaluation. Previously a homozygous 163C-T transition in exon 2 of the TUSC3 gene, resulting in a gln55-to-ter (Q55X) substitution has been reported. And here we predicted by PHD-SNP that X could be either proline (P) or Aspartic acid (D) causing deleterious effect. As mutation at 55 position was responsible for non-syndromic autosomal recessive mental retardation, so it was assumed that mutation was either by proline or aspartic acid. Further, MUPRO, PROVEAN, I-Mutant, Predict SNP, Meta SNP, Panther, SIFT, SNPs 3D confirmed that deleterious mutation was Q55P transition... .

Effects of p35 Mutations Associated with Mental Retardation on the Cellular Function of p35-CDK5

p35 is an activation subunit of the cyclin-dependent kinase 5 (CDK5), which is a Ser/Thr kinase that is expressed predominantly in neurons. Disruption of the CDK5 or p35 (CDK5R1) genes induces abnormal neuronal layering in various regions of the mouse brain via impaired neuronal migration, which may be relevant for mental retardation in humans. Accordingly, mutations in the p35 gene were reported in patients with nonsyndromic mental retardation; however, their effect on the biochemical function of p35 has not been examined. Here, we studied the biochemical effect of mutant p35 on its known properties, i.e., stability, CDK5 activation, and cellular localization, using heterologous expression in cultured cells. We also examined the effect of the mutations on axon elongation in cultured primary neurons and migration of newborn neurons in embryonic brains. However, we did not detect any significant differences in the effects of the mutant forms of p35 compared with wild-type p35. Therefore, we conclude that these p35 mutations are unlikely to cause mental retardation.

Syndromic craniosynostosis associated with microdeletion of chromosome 19p13.12–19p13.2

Craniosynostosis, a condition in which the cranial sutures prematurely fuse, can lead to elevated intracranial pressure and craniofacial abnormalities in young children. Currently surgical intervention is the only therapeutic option for patients with this condition. Craniosynostosis has been associated with a variety of different gene mutations and chromosome anomalies. Here we describe three cases of partial deletion of chromosome 19p. Two of the cases present with syndromic craniosynostosis while one has metopic ridging. A review of the genes involved in the rearrangements between the three cases suggests several gene candidates for craniosynostosis. CALR and DAND5, BMP regulators involved in osteoblast differentiation, and MORG1, a mediator of osteoclast dysregulation may play a role in abnormal cranial vault development. Additionally, CACNA1A, a gene that when mutated is associated with epilepsy and CC2D1A, a gene associated with non-syndromic mental retardation may contribute to additional phenotypic features seen in the patients we describe. In addition, these findings further support the need for genetic testing in cases of syndromic craniosynostosis.

Cytogenetic analysis in a large series of children with non-syndromic mental retardation.

Mental retardation affects 1-3% of the population. To evaluate the implication of chromosomal abnormalities in the etiology of mental retardation, 1420 patients with non-syndromic mental retardation recruited at the department of cytogenetics of Farhat Hached hospital (Sousse, Tunisia) between January 2005 and December 2009, were analyzed using standard cytogenetic techniques. Age ranged between 3 and 18 years with a median of 8 years. Chromosomal abnormalities were detected in 7.8% of patients and an increased prevalence of chromosome anomalies was observed in patients when the mental retardation is associated with a severe degree of intellectual disability, facial dysmorphic features and/or congenital malformations or epilepsy.

Association of TUSC3 gene polymorphisms with non-syndromic mental retardation based on nuclear families in the Qinba mountain area of China.

TUSC3 interacts with the protein phosphatase 1 and magnesium ion transport system, which plays an important role in learning and memory. Abnormal conditions of learning and memory are common clinical characteristics of mental retardation (MR). However, the association of TUSC3 genetic polymorphisms with MR remains unknown. A total of 456 DNA samples including 174 nuclear families containing MR were collected in the Qinba mountain area of China. The genotypes of eight tag single nucleotide polymorphisms of TUSC3 were evaluated with traditional genetic methods. Family-based association tests, transmission disequilibrium tests (TDTs), and haplotype relative risk (HRR) analyses were performed to investigate the association between genetic variants of the TUSC3 gene and MR. The genetic polymorphisms rs10093881, rs6530893, and rs6994908 were associated with MR (all P values <0.05) based upon the results of single-site TDT and HRR analyses. The haplotype block consisting of rs6530893 and rs6994908, harboring the sixth exon of TUSC3, was also associated with MR (all P values <0.05). This study demonstrated an association between genetic polymorphisms of the TUSC3 gene and MR in the Qinba mountain area, the sixth exon of which might contribute to the risk of MR. However, further studies are needed on the causal mechanisms in this association.

Inactivition of CDKL3 mildly inhibits proliferation of cells at VZ/SVZ in brain.

CDKL3 has an important role in regulating cell growth and/or differentiation, and its inactivation is recently reported to be related to non-syndromic mild mental retardation (MR). MR is a common neurological disorder, predominantly characterized by impaired cognitive function. Though genetic factors play a very important role in the pathogenesis of MR, to date, only few genes linked to MR have been characterized and understood very well. Here, we investigated the role of the CDKL3 in the proliferation of cells surrounding the brain ventricle, and the results showed down-regulating CDKL3 by the method of RNAi in the cells surrounding the brain ventricle of the mouse embryo at E15 may inhibit their proliferation. As our previous study had shown that Cdkl3 mRNA expression is developmentally regulated in the central nervous system, peaking during late embryonic and early postnatal stages which are the key stages of neurite formation and maturation, furtherly, the present findings indicated that CDKL3 may be involved in proliferation of cells surrounding the brain ventricle where neuronal progenitor cells are enriched during the late embryo stage, supporting the notion that CDKL3 inactivation contributes to non-syndromic mild MR.

Highly Expressed Genes within Hippocampal Sector CA1: Implications for the Physiology of Memory.

As the CA1 sector has been implicated to play a key role in memory formation, a dedicated search for highly expressed genes within this region was made from an on-line atlas of gene expression within the mouse brain (GENSAT). From a data base of 1013 genes, 16 were identified that had selective localization of gene expression within the CA1 region, and included Angpt2, ARHGEF6, CCK, Cntnap1, DRD3, EMP1, Epha2, Itm2b, Lrrtm2, Mdk, PNMT, Ppm1e, Ppp2r2d, RASGRP1, Slitrk5, and Sstr4. Of the 16 identified, the most selective and intense localization for both adult and post-natal day 7 was noted for ARHGEF6, which is known to be linked to non-syndromic mental retardation, and has also been localized to dendritic spines. Further research on the role played by ARHGEF6 in memory formation is strongly advocated

Structural Basis of Substrate Specificity of Human Oligosaccharyl Transferase Subunit N33/Tusc3 and Its Role in Regulating Protein N-Glycosylation

N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is essential in eukaryotes and catalyzed by oligosaccharyl transferase (OST). Human OST is a hetero-oligomer of seven subunits. The subunit N33/Tusc3 is a tumor suppressor candidate, and defects in the subunit N33/Tusc3 are linked with nonsyndromic mental retardation. Here, we show that N33/Tusc3 possesses a membrane-anchored N-terminal thioredoxin domain located in the ER lumen that may form transient mixed disulfide complexes with OST substrates. X-ray structures of complexes between N33/Tusc3 and two different peptides as model substrates reveal a defined peptide-binding groove adjacent to the active site that can accommodate peptides in opposite orientations. Structural and biochemical data show that N33/Tusc3 prefers peptides bearing a hydrophobic residue two residues away from the cysteine forming the mixed disulfide with N33/Tusc3. Our results support a model in which N33/Tusc3 increases glycosylation efficiency for a subset of human glycoproteins by slowing glycoprotein folding.